Methods of treating congenital hemophilia with anti-fibrinolytic loaded platelets

ABSTRACT

Provided herein are methods and compositions for treating congenital hemophilia with platelets and/or platelet derivatives. In some cases, the platelets and or platelet derivatives are loaded with an anti-fibrinolytic.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/969,942, filed on Feb. 4, 2020, U.S. Provisional Patent Application No. 62/980,850, filed on Feb. 24, 2020, and U.S. Provisional Patent Application No. 63/065,337, filed on Aug. 13, 2020, the contents of which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

Provided herein are compositions and methods for use of platelets, platelet derivatives, or thrombosomes (e.g., freeze-dried platelet derivatives) as biological carriers of cargo, such as anti-fibrinolytic compounds, also referred to herein as anti-fibrinolytic loaded platelets, platelet derivatives, or thrombosomes.

BACKGROUND

Blood is a complex mixture of numerous components. In general, blood can be described as comprising four main parts: red blood cells, white blood cells, platelets, and plasma. The first three are cellular or cell-like components, whereas the fourth (plasma) is a liquid component comprising a wide and variable mixture of salts, proteins, and other factors necessary for numerous bodily functions. The components of blood can be separated from each other by various methods. In general, differential centrifugation is most commonly used currently to separate the different components of blood based on size and, in some applications, density.

Unactivated platelets, which are also commonly referred to as thrombocytes, are small, often irregularly-shaped (e.g., discoidal or ovoidal) megakaryocyte-derived components of blood that are involved in the clotting process. They aid in protecting the body from excessive blood loss due not only to trauma or injury, but to normal physiological activity as well.

Platelets are considered crucial in normal hemostasis, providing the first line of defense against blood escaping from injured blood vessels. Platelets generally function by adhering to the lining of broken blood vessels, in the process becoming activated, changing to an amorphous shape, and interacting with components of the clotting system that are present in plasma or are released by the platelets themselves or other components of the blood. Purified platelets have found use in treating subjects with low platelet count (thrombocytopenia) and abnormal platelet function (thrombasthenia). Concentrated platelets are often used to control bleeding after injury or during acquired platelet function defects or deficiencies, for example those occurring during surgery and those due to the presence of platelet inhibitors.

SUMMARY OF THE INVENTION

Platelet transfusion and anti-fibrinolytics are suitable for improving traumatic bleeding events (e.g., hemorrhage). Methods and compositions provided herein generally describe the use of platelets as biological carriers of anti-fibrinolytic compounds, including but not limited to, F-aminocaproic acid (EACA). Loading anti-fibrinolytics into platelets can shield the anti-fibrinolytic from systemic exposure and metabolic processes as the platelet migrates to the site of injury. At the site of injury the drug can be released to enhance the stability of the forming clot. Anti-fibrinolytic (e.g., EACA) loaded platelets can be cryopreserved for long term storage, can retain the internalized cargo (e.g., EACA) after thawing, and can release the anti-fibrinolytic in response to in vitro stimulation by endogenous platelet agonists.

Provided herein are methods and compositions that are suitable for improving traumatic bleeding (e.g., hemorrhage) therapy by reducing the therapeutic dose of anti-fibrinolytic and prevent unwanted side effects as a result of off-site drug interactions. Also provided herein are methods and compositions that are used to treat hemophilia, including classic (e.g., inherited) hemophilia with anti-fibrinolytic (e.g., EACA) loaded platelets. In another aspect methods and compositions that can be used to treat hemophilia, including classic (e.g., inherited) hemophilia with thrombosomes (e.g., unloaded thrombosomes).

Provided herein are methods of treating a coagulopathy in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a coagulopathy in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of restoring normal hemostasis in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of restoring normal hemostasis in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of preparing a subject for surgery, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of preparing a subject for surgery, the method comprising administering to the subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemorrhage in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemorrhage in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemophilia in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemophilia in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition. In some embodiments, the platelets or platelet derivatives can be loaded with an anti-fibrinolytic.

Provided herein are methods of treating a hemorrhage in a subject, wherein the subject is a subject in need thereof, including administering a therapeutically effective amount of anti-fibrinolytic loaded platelets to the subject in need thereof.

Provided herein are methods of treating classic hemophilia A in a subject, wherein the subject is a subject in need thereof, including administering a therapeutically effective amount of anti-fibrinolytic loaded platelets to the subject in need thereof.

Provided herein are methods of treating classic hemophilia B in a subject, wherein the subject is a subject in need thereof, including administering a therapeutically effective amount of anti-fibrinolytic loaded platelets to the subject in need thereof.

In some embodiments of any of the methods provided herein, the concentration of the therapeutically effective amount of anti-fibrinolytic loaded into the platelets is from about 100 μM to about 10 mM.

Also provided herein are methods of treating classic hemophilia A in a subject, wherein the subject is a subject in need thereof, including administering a therapeutically effective amount of unloaded thrombosomes to the subject in need thereof.

Provided herein are methods of treating classic hemophilia B in a subject, wherein the subject is a subject in need thereof, including administering a therapeutically effective amount of unloaded thrombosomes to the subject in need thereof.

In some embodiments of administering a therapeutically effective amount of unloaded thrombosomes to a subject in thereof, includes a concentration of the therapeutically effective amount of unloaded thrombosomes from about 1×10² particles/kg to about 1×10¹³ particles/kg.

In another aspect provided herein are methods of preparing anti-fibrinolytic loaded platelets including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets. In some embodiments (e.g., for unloaded platelets or platelet derivatives), a “loading buffer” may be alternatively called an “incubating agent”.

Provided herein are methods of preparing anti-fibrinolytic loaded platelets, including providing platelets and contacting the platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are contacted with the anti-fibrinolytic and with the loading buffer sequentially, in either order.

Provided herein are methods of preparing anti-fibrinolytic loaded platelets, including contacting platelets with the anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods of preparing anti-fibrinolytic loaded platelets, including contacting the platelets with a buffer including a salt, a base, a loading agent, and optionally at least one organic solvent to form a first composition and contacting the first composition with an anti-fibrinolytic, to form the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are contacted with the anti-fibrinolytic and with the loading buffer concurrently.

In another aspect provided herein are methods of preparing anti-fibrinolytic loaded platelets, including contacting the platelets with an anti-fibrinolytic in the presence of a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent to form the drug-loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded, the platelets are pooled from a plurality of donors prior to a treating step.

Provided herein are methods of preparing anti-fibrinolytic loaded platelets, including A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods of preparing anti-fibrinolytic loaded platelets, including A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods of preparing anti-fibrinolytic loaded platelets, including A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition and contacting the first composition with an anti-fibrinolytic to form the anti-fibrinolytic loaded platelets.

Also provided herein are methods of preparing anti-fibrinolytic loaded platelets, including A) pooling platelets from a plurality of donors and B) contacting the platelets with a drug in the presence of a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the loading agent is a monosaccharide or a disaccharide. In some embodiments of preparing anti-fibrinolytic loaded platelets, the loading agent is sucrose, maltose, trehalose, glucose, mannose, or xylose. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are isolated prior to a contacting step. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are loaded with the drug in a period of time of 5 minutes to 48 hours. In some embodiments of preparing anti-fibrinolytic loaded platelets, the concentration of the anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 1 μM to about 100 mM. In some embodiments of preparing anti-fibrinolytic loaded platelets, the one or more organic solvents is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof. In some embodiments of preparing anti-fibrinolytic loaded platelets, cold storing, cryopreserving, freeze-drying, thawing, rehydrating, and combinations thereof the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the drying step includes freeze-drying the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, further including rehydrating the anti-fibrinolytic loaded platelets obtained from the drying step.

Also provided herein are anti-fibrinolytic loaded platelets prepared by any of the methods described herein.

Also provided herein are rehydrated anti-fibrinolytic loaded platelets prepared by a method including rehydrating the anti-fibrinolytic loaded platelets by any of the methods described herein. In some embodiments of preparing anti-fibrinolytic loaded platelets, the anti-fibrinolytic is modified with an imaging agent. In some embodiments of preparing anti-fibrinolytic loaded platelets, the anti-fibrinolytic is modified with the imaging agent prior to contacting platelets with the anti-fibrinolytic. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are further treated with an imaging agent, where the anti-fibrinolytic loaded platelets are loaded with the imaging agent. In some embodiments of preparing anti-fibrinolytic loaded platelets, the method does not include contacting the platelets with an organic solvent. In some embodiments of preparing anti-fibrinolytic loaded platelets, the method does not include contacting the first composition with an organic solvent. In some embodiments of preparing anti-fibrinolytic loaded platelets, the anti-fibrinolytic is selected from the group consisting of ε-aminocaproic acid, aprotinin, aminomethylbenzoic acid, tranexamic acid, and fibrinogen. In some embodiments of preparing anti-fibrinolytic loaded platelets, the F-aminocaproic acid is present in a concentration of at least 100 μM.

Provided herein are anti-fibrinolytic loaded platelets prepared by any of the methods described herein.

Also provided herein are methods where the anti-fibrinolytic-loaded platelets treat a hemorrhage in a subject, wherein the subject is a subject in need thereof.

Also provided herein are methods where the anti-fibrinolytic loaded platelets treat a disease in a subject wherein the subject is a subject in need thereof. In some embodiments where the anti-fibrinolytic loaded platelets treat a disease, the anti-fibrinolytic loaded platelets treat hemophilia. In some embodiments, the hemophilia is classic hemophilia.

Also provided herein, are methods of preparing platelets, platelet derivatives, or thrombosomes loaded with anti-fibrinolytic compounds. Also provided herein, are methods of treating conditions, such as hemophilia, or conditions such as hemorrhaging with platelets, platelet derivatives, thrombosomes, and/or thrombosomes loaded with anti-fibrinolytic compounds. Also provided herein, are methods of treating conditions such as congenital hemophilia, or conditions such as hemorrhaging (e.g., trauma) with unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes.

Also provided herein are methods and compositions that are suitable for treating drug-induced coagulopathy, such as antiplatelet agent-induced coagulopathy, such as, for example, treatment with anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded thrombosomes, or anti-fibrinolytic loaded platelet derivatives.

Also provided herein are methods and compositions that are suitable for treating coagulopathy, such as a disease-caused coagulopathy (e.g., congenital hemophilia) or a drug-induced coagulopathy, such as, for example, treatment with anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded thrombosomes, or anti-fibrinolytic loaded platelet derivatives.

Also provided herein are methods and compositions that are suitable for treating drug-induced coagulopathy, such as, for example, treatment with platelets, thrombosomes, or platelet derivatives.

Also provided herein are methods and compositions that are suitable for treating coagulopathy, such as antiplatelet agent-induced coagulopathy, such as for example, treatment with platelets, thrombosomes, or platelet derivatives.

Anti-fibrinolytic loaded platelets described herein can be stored under typical ambient conditions, refrigerated, cryopreserved, for example with dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization (e.g., to form thrombosomes)

Also provided herein are methods of treating a hemorrhage in a subject, wherein the subject is a subject in need thereof, including administering a therapeutically effective amount of unloaded thrombosomes to the subject in need thereof. In some embodiments of treating a hemorrhage in a subject, wherein the subject is a subject in need thereof, the concentration of the therapeutically effective amount of unloaded thrombosomes is from about 1×10² particles/kg to about 1×10¹³ particles/kg.

Also provided herein are methods of treating congenital Hemophilia A in a subject, wherein the subject is a subject in need thereof, the method including, administering a therapeutically effective amount of loaded thrombosomes to a subject in need thereof, wherein the loaded thrombosomes are loaded with an anti-fibrinolytic.

Also provided herein are methods of treating congenital Hemophilia B in a subject, wherein the subject is a subject in need thereof, the method including, administering a therapeutically effective amount of loaded thrombosomes to a subject in need thereof, wherein the loaded thrombosomes are loaded with an anti-fibrinolytic.

In some embodiments of any of the methods described herein, the anti-fibrinolytic is selected from the group including of ε-aminocaproic acid, aprotinin, aminomethylbenzoic acid, tranexamic acid, and fibrinogen.

Also, provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition, where the subject has been treated or is being treated with an anticoagulant.

Also, provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including anti-fibrinolytic loaded platelets, where the subject has been treated or is being treated with an anticoagulant.

Also, provided herein are methods of restoring normal hemostasis in a subject, the method including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition, where the subject has been treated or is being treated with an anticoagulant.

Also, provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition including anti-fibrinolytic loaded platelets, where the subject has been treated or is being treated with an anticoagulant.

Also, provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof an effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition, where the subject has been treated or is being treated with an anticoagulant.

In some embodiments of preparing a subject for surgery, the surgery is an emergency surgery. In some embodiments of preparing a subject for surgery the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, treatment with the anticoagulant is stopped. In some embodiments of any of the methods described herein, treatment with the anticoagulant is continued.

Also provided herein are methods of ameliorating the effects of an anticoagulant in a subject, including administering to the subject in need thereof a therapeutically effective amount of anti-fibrinolytic loaded platelets.

Also provided herein are methods of ameliorating the effects of an anticoagulant in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some embodiments of ameliorating the effects of anticoagulant in a subject, the composition is administered following administration to the subject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, a low molecular weight heparin, a supplement, and a combination thereof.

In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, low molecular weight heparins, tifacogin, Factor VIIai, SB249417, pegnivacogin (with or without anivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon, acenocoumarol, indandiones, fluindione, and a supplement, and a combination thereof.

In some embodiments of any of the methods described herein, the anticoagulant is warfarin. In some embodiments of any of the methods described herein, the anticoagulant is heparin.

In some embodiments of any of the methods described herein, the method includes drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition following the drying step.

In some embodiments of any of the methods described herein, the method includes freeze-drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition following the freeze-drying step.

In some embodiments of any of the methods described herein, one or more organic solvents is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof. In some embodiments of any of the methods described herein, the composition includes an organic solvent.

In some embodiments of any of the methods described herein, the anti-fibrinolytic loaded platelets or anti-fibrinolytic loaded platelet derivatives includes thrombosomes.

Also provided here are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including anti-fibrinolytic loaded platelets or anti-fibrinolytic loaded platelet derivatives and a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided here are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided here are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including anti-fibrinolytic loaded platelets or anti-fibrinolytic loaded platelet derivatives and a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided here are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided here are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition including anti-fibrinolytic loaded platelets or anti-fibrinolytic loaded platelet derivatives and a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided here are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition, where the subject has been treated or is being treated with an antiplatelet agent.

In some embodiments of preparing a subject for surgery, the surgery is an emergency surgery. In some embodiments of preparing a subject for surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, treatment with the antiplatelet agent is stopped. In some embodiments of any of the methods described herein, treatment with the antiplatelet agent is continued.

Also provided herein are methods of ameliorating the effects of an antiplatelet agent in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including anti-fibrinolytic loaded platelets or anti-fibrinolytic loaded platelet derivatives and a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent.

Also provided herein are methods of ameliorating the effects of an antiplatelet agent in a subject, the method including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some embodiments of ameliorating the effects of an antiplatelet agent in a subject, the composition is administered following administration to the subject or assumption by subject, or an overdose of the antiplatelet agent.

In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, and a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, and atopaxar, and a combination thereof. In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate, and a combination thereof.

In some embodiments of any of the methods described herein, the method includes drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition following the drying step. In some embodiments of any of the methods described herein, the method includes freeze-drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition following the freeze-drying step.

In some embodiments of any of the methods described herein, one or more organic solvents is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof. In some embodiments of any of the methods described herein, the composition includes an organic solvent.

Also provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, where the subject has been treated or is being treated with an anticoagulant.

Also provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition, where the subject has been treated or is being treated with an anticoagulant.

Also provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, where the subject has been treated or is being treated with an anticoagulant.

Also provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition, where the subject has been treated or is being treated with an anticoagulant.

Also provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, where the subject has been treated or is being treated with an anticoagulant.

Also provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition, where the subject has been treated or is being treated with an anticoagulant.

In some embodiments of preparing a subject for surgery, the surgery is an emergency surgery. In some embodiments of preparing a subject for surgery, the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, the subject or is being treated with an anticoagulant. In some embodiments of any of the methods described herein, treatment with the anticoagulant is stopped. In some embodiments of any of the methods described herein, treatment with the anticoagulant is continued.

Also provided herein are methods of ameliorating the effects of an anticoagulant in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of ameliorating the effects of an anticoagulant in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

In some embodiments of ameliorating the effects of an anticoagulant in a subject, the composition is administered following administration to the subject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments of any of the methods described herein, the composition includes an anti-fibrinolytic agent. In some embodiments of any of the methods described herein, the anti-fibrinolytic agent is selected from the group consisting of F-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and a combination thereof.

In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, a low molecular weight heparin, and a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, low molecular weight heparins, tifacogin, Factor VIIai, SB249417, pegnivacogin (with or without anivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon, acenocoumarol, indandiones, fluindione, and a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the anticoagulant is warfarin. In some embodiments of any of the methods described herein, the anticoagulant is heparin.

In some embodiments of any of the methods described herein, before administering, the subject had an INR of at least 4.0. In some embodiments of any of the methods described herein, after the administering, the subject has an INR of 3.0 or less. In some embodiments of any of the methods described herein, after the administering, the subject has an INR of 2.0 or less. In some embodiments of any of the methods described herein, before administering, the subject had an INR of at least 3.0. In some embodiments of any of the methods described herein, after administering, the subject has an INR of 2.0 or less.

In some embodiments of any of the methods described herein, administering includes administering topically. In some embodiments of any of the methods described herein, administering includes administering parenterally. In some embodiments of any of the methods described herein, administering includes administering intravenously. In some embodiments of any of the methods described herein, administering includes administering intramuscularly. In some embodiments of any of the methods described herein, administering includes administering intrathecally. In some embodiments of any of the methods described herein, administering includes administering subcutaneously. In some embodiments of any of the methods described herein, administering includes administering intraperitoneally.

In some embodiments of any of the methods described herein, the method includes drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition is following the drying step. In some embodiments of any of the methods described herein, the method includes freeze-drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition following the freeze-drying step.

In some embodiments of any of the methods described herein, the incubating agent includes one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and a combination of two or more thereof.

In some embodiments of any of the methods described herein, the incubating agent includes a carrier protein. In some embodiments of any of the methods described herein, the buffer includes HEPES, sodium bicarbonate (NaHCO₃), or a combination thereof. In some embodiments of any of the methods described herein, the composition includes one or more saccharides. In some embodiments of any of the methods described herein, the one or more saccharides includes trehalose. In some embodiments of any of the methods described herein, the one or more saccharides includes polysucrose. In some embodiments of any of the methods described herein, the one or more saccharides includes dextrose.

In some embodiments of any of the methods described herein, the composition includes an organic solvent.

In some embodiments of any of the methods described herein, the platelets or platelet derivatives includes thrombosomes.

Also provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, where the subject has been treated or is being treated with an antiplatelet agent.

Also provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition, where the subject has been treated or is being treated with an antiplatelet agent.

In some embodiments of preparing a subject for surgery, the surgery is an emergency surgery. In some embodiments of preparing a subject for surgery, the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, treatment with the antiplatelet agent is stopped. In some embodiments of any of the methods described herein, treatment with the antiplatelet agent is continued.

Also provided herein are methods of ameliorating the effects of an antiplatelet agent in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of ameliorating the effects of an antiplatelet agent in a subject, including administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

In some embodiments of ameliorating the effects of an antiplatelet agent in a subject, the composition is administered following administration to the subject or assumption by subject, or an overdose of the antiplatelet agent.

In some embodiments of any of the methods described herein, the composition includes an anti-fibrinolytic agent. In some embodiments of any of the methods described herein, the anti-fibrinolytic agent is selected from the group consisting of F-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and a combination thereof.

In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, and a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, and atopaxar, and a combination thereof. In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate, and a combination thereof.

In some embodiments of any of the methods described herein, administering includes administering topically. In some embodiments of any of the methods described herein, administering includes administering parenterally. In some embodiments of any of the methods described herein, administering includes administering intravenously. In some embodiments of any of the methods described herein, where administering includes administering intramuscularly. In some embodiments of any of the methods described herein, where administering includes administering intrathecally. In some embodiments of any of the methods described herein, where administering includes administering subcutaneously. In some embodiments of any of the methods described herein, where administering includes administering intraperitoneally.

In some embodiments of any of the methods described herein, the method includes drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method includes rehydrating the composition after the drying step. In some embodiments of any of the methods described herein, the method includes freeze-drying the composition prior to the administration step. In some embodiments of any of the methods described herein, the method rehydrating the composition is after the freeze-drying step.

In some embodiments of any of the methods described herein, the incubating agent includes one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and a combination of two or more thereof. In some embodiments of any of the methods described herein, where the incubating agent includes a carrier protein. In some embodiments of any of the methods described herein, the buffer includes HEPES, sodium bicarbonate (NaHCO₃), or a combination thereof.

In some embodiments of any of the methods described herein, where the composition includes one or more saccharides. In some embodiments of any of the methods described herein, one or more saccharides includes trehalose. In some embodiments of any of the methods described herein, the one or more saccharides includes polysucrose. In some embodiments of any of the methods described herein, the one or more saccharides includes dextrose.

In some embodiments of any of the methods described herein, the composition includes an organic solvent.

In some embodiments of any of the methods described herein, the platelets or platelet derivatives includes thrombosomes.

Also provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof an effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of treating a coagulopathy in a subject, including administering to the subject in need thereof an effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

In some embodiments of treating a coagulopathy in a subject, the composition is administered following administration to the subject an antiplatelet agent or an anticoagulant, or a subject having hemophilia.

Also provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof an effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of restoring normal hemostasis in a subject, including administering to the subject in need thereof an effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

Also provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof an effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of preparing a subject for surgery, including administering to the subject in need thereof an effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

In some embodiments of preparing a subject for surgery, the surgery is an emergency surgery.

In some embodiments of preparing a subject for surgery, the surgery is a scheduled surgery.

In some embodiments of any of the methods described herein, the subject has been treated or is being treated with an anticoagulant. In some embodiments of any of the methods described herein, treatment with the anticoagulant is stopped. In some embodiments of any of the methods described herein, treatment with the anticoagulant is continued.

Also provided herein are methods of ameliorating the effects of an anticoagulant in a subject, including administering to the subject in need thereof an effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of ameliorating the effects of an anticoagulant in a subject, including administering to the subject in need thereof an effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

In some embodiments of ameliorating the effects of an anticoagulant in a subject, the composition is administered following administration to the subject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, a low molecular weight heparin, a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, low molecular weight heparins, tifacogin, Factor VIIai, SB249417, pegnivacogin (with or without anivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon, acenocoumarol, indandiones, fluindione, a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the anticoagulant is warfarin. In some embodiments of any of the methods described herein, the anticoagulant is heparin.

In some embodiments of any of the methods described herein, before the administering, the subject had an INR of at least 4.0. In some embodiments of any of the methods described herein, after the administering, the subject has an INR of 3.0 or less. In some embodiments of any of the methods described herein, after the administering, the subject has an INR of 2.0 or less. In some embodiments of any of the methods described herein, before the administering, the subject had an INR of at least 3.0. In some embodiments of any of the methods described herein, after the administering, the subject has an INR of 2.0 or less.

In some embodiments of any of the methods described herein, the subject has been treated or is being treated with an anti-platelet agent. In some embodiments of any of the methods described herein, treatment with the antiplatelet agent is stopped. In some embodiments of any of the methods described herein, treatment with the antiplatelet agent is continued.

Also provided herein are methods of ameliorating the effects of an antiplatelet agent in a subject, including administering to the subject in need thereof an effective amount of a composition including platelets or platelet derivatives and an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Also provided herein are methods of ameliorating the effects of an antiplatelet agent in a subject, including administering to the subject in need thereof an effective amount of a composition prepared by a process including incubating platelets with an incubating agent including one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

In some embodiments of ameliorating the effects of an antiplatelet agent in a subject, the composition is administered following administration to the subject or assumption by subject, or an overdose of the antiplatelet agent.

In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, a supplement, and a combination thereof. In some embodiments of any of the methods described herein, the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, and a combination thereof. In some embodiments of any of the methods described herein, where the antiplatelet agent is selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, sarpogrelate, and a combination thereof.

In some embodiments of any of the methods described herein, the composition includes an anti-fibrinolytic agent. In some embodiments of any of the methods described herein, the anti-fibrinolytic agent is selected from the group consisting of F-aminocaproic acid (EACA), tranexamic acid, aprotinin, aminomethylbenzoic acid, fibrinogen, and a combination thereof. In some embodiments of any of the methods described herein, the platelets or platelet derivatives are loaded with the anti-fibrinolytic agent.

In some embodiments of any of the methods described herein, administering includes administering topically. In some embodiments of any of the methods described herein, administering includes administering parenterally. In some embodiments of any of the methods described herein, administering includes administering intravenously. In some embodiments of any of the methods described herein, administering includes administering intramuscularly. In some embodiments of any of the methods described herein, administering includes administering intrathecally. In some embodiments of any of the methods described herein, administering includes administering subcutaneously. In some embodiments of any of the methods described herein, administering includes administering intraperitoneally.

In some embodiments, the anti-fibrinolytic is F-aminocaproic acid. In some embodiments, the ε-aminocaproic acid is present in a concentration from about 1 μM to about 100 mM.

In some embodiments of any of the methods described herein, the incubating agent includes a carrier protein. In some embodiments of any of the methods described herein, the buffer includes HEPES, sodium bicarbonate (NaHCO₃), or a combination thereof. In some embodiments of any of the methods described herein, the composition includes one or more saccharides. In some embodiments of any of the methods described herein, the one or more saccharides includes trehalose. In some embodiments of any of the methods described herein, the one or more saccharides includes polysucrose. In some embodiments of any of the methods described herein, the one or more saccharides includes dextrose.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing dose-dependent ε-aminocaproic acid (EACA) loading into platelets at 50 mM and 100 mM using Dansyl-EACA at a molar ratio of 1:1000 in the loading buffer in order to measure loading via fluorescence over time at 1 hour, 2 hours, 3 hours, and 4 hours.

FIG. 2 is a graph showing in vitro agonist stimulation of EACA release from EACA-loaded platelets with PMA, collagen, and TRAP agonists using Dansyl-EACA at a molar ratio of 1:1000 in the loading buffer in order to measure release via fluorescence.

FIG. 3 is a graph showing an EACA dose-response curve in pooled human platelet rich plasma to determine the effect of free EACA on lysis after 30 minutes (LY30) via thromboelastography.

FIGS. 4A-4E show that EACA-loaded platelets can release EACA in vitro to prevent fibrinolysis. FIGS. 4A-E show thromboelastogram (TEG) graphs of EACA loaded platelets with tissue plasminogen activator at varying platelet concentrations. FIG. 4F shows a dose-response curve of the effect of EACA-loaded platelets on LY30.

FIG. 5 is a graph comparing the percent lysis of clots at 30 minutes for free EACA in solution (FIG. 3) and EACA-loaded platelets (FIGS. 4A-E) showing improved response from EACA loaded into platelets.

FIG. 6 is a graph measuring the amount of EACA mg/platelet of EACA-loaded platelets pre-cryopreservation and post-cryopreservation using Dansyl-EACA at a molar ratio of 1:1000 in the loading buffer in order to measure loading via fluorescence.

FIGS. 7A-7E that cryopreserved EACA-loaded platelets can release EACA in vitro to prevent fibrinolysis. FIGS. 7A-D show TEG graphs of cryopreserved EACA loaded platelets plus tissue plasminogen activator at varying platelet concentrations. FIG. 7E shows a dose-response curve of cryopreserved EACA loaded platelets.

FIGS. 8A-C show graphs indicating the strength of clots as measured by maximum amplitude (MA). FIG. 8A shows MA measured in the presence of free EACA.

FIGS. 8B-C shows MA measured with EACA loaded platelets pre-cryopreservation (8B) and post-cryopreservation (8C).

FIG. 9 is a histogram showing thrombin production after clot initiation in George King plasma, 95% Factor IX deficient plasma, and 95% Factor IX deficient plasma plus thrombosomes.

FIG. 10 is a graph showing the depression of thrombin production in Factor IX deficient plasma is partially recovered by thrombosomes.

FIG. 11 is a graph showing the loss of endogenous thrombin potential (ETP) in Factor IX deficient plasma is partially recovered by thrombosomes.

FIGS. 12A-E show a thromboelastography graphs measuring hemostasis in a platelet rich plasma model of congenital Hemophilia A under various conditions.

FIG. 13 is a graph showing thrombin generation (nM) under various conditions in 100% immunodepleted Factor IX (Hemophilia B) plasma over time.

FIG. 14 is a graph showing thrombin generation (nM) under various conditions in 99% immunodepleted Factor IX (Hemophilia B) plasma over time.

FIG. 15 is a graph showing thrombin generation (nM) under various conditions in 90% immunodepleted Factor IX (Hemophilia B) plasma over time.

FIG. 16 is a graph showing thrombin generation (peak thrombin) in a platelet rich plasma model of Hemophilia B under various conditions over time.

FIG. 17 is a graph showing restoration of thrombin generation (total thrombin) in a platelet rich plasma model of Hemophilia B under various conditions.

FIG. 18 is a graph showing restoration of thrombin generation (nM) in congenital Factor VIII deficient (Hemophilia A) plasma rich platelets under various conditions.

FIG. 19 is a graph showing restoration of thrombin generation (nM) in congenital Factor IX deficient (Hemophilia B) plasma rich platelets under various conditions.

FIG. 20 is a graph showing restoration of peak thrombin generation (nM) in congenital Factor VIII (Hemophilia A) and Factor IX (Hemophilia B) deficient platelet rich plasma under various conditions.

FIG. 21 is graph showing restoration of thrombin generation (total thrombin) in congenital Factor VIII (Hemophilia A) and Factor IX (Hemophilia B) deficient platelet rich plasma under various conditions.

FIG. 22 is a graph showing titration of EACA-loaded thrombosomes (nM) in George King pooled plasma (“normal”).

FIG. 23 is a graph showing titration of EACA-loaded thrombosomes (nM) in 95% congenital Factor IX (Hemophilia B) deficient plasma.

FIG. 24 is graph showing restoration of thrombin generation (peak thrombin) in 95% congenital Factor IX (Hemophilia B) under various conditions.

FIG. 25 is a graph showing restoration of thrombin generation (total) in 95% Factor IX deficient plasma under various conditions.

FIG. 26 is a graph showing restoration of thrombin generation (total) in 95% Factor IX deficient plasma under various conditions.

FIG. 27 is a graph showing titration of EACA-loaded thrombosomes in congenital Factor VIII (Hemophilia A) deficient plasma.

FIG. 28 is a graph showing restoration of thrombin generation (nM) in platelet rich plasma models of congenital Hemophilia A.

DETAILED DESCRIPTION

This disclosure is directed to compositions and methods for use of platelets, platelet derivatives, or thrombosomes as biological carriers of cargo, such as anti-fibrinolytic compounds, also referred to herein as anti-fibrinolytic loaded platelets, platelet derivatives, or thrombosomes. Also provided herein, are methods of preparing platelets, platelet derivatives, or thrombosomes loaded with anti-fibrinolytic compounds. This disclosure is also directed to compositions and methods for use of unloaded platelets, platelet derivatives, or thrombosomes in the treatment of a disease such as congenital hemophilia, or conditions such as hemorrhaging or trauma.

Anti-fibrinolytic loaded platelets described herein can be stored under typical ambient conditions, refrigerated, cryopreserved, for example with dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization (e.g., to form thrombosomes).

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Further, where a range of values is disclosed, the skilled artisan will understand that all other specific values within the disclosed range are inherently disclosed by these values and the ranges they represent without the need to disclose each specific value or range herein. For example, a disclosed range of 1-10 includes 1-9, 1-5, 2-10, 3.1-6, 1, 2, 3, 4, 5, and so forth. In addition, each disclosed range includes up to 5% lower for the lower value of the range and up to 5% higher for the higher value of the range. For example, a disclosed range of 4-10 includes 3.8-10.5. This concept is captured in this document by the term “about”.

As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a platelet” includes a plurality of such platelets. Furthermore, the use of terms that can be described using equivalent terms include the use of those equivalent terms. Thus, for example, the use of the term “subject” is to be understood to include the terms “patient”, “individual,” or “animal” and other terms used in the art to indicate one who is subject to a treatment.

As used herein, and unless otherwise specified, the terms “treat,” “treating” and “treatment” contemplate an action that occurs while a subject is suffering from a disease (e.g., congenital hemophilia), disorder, and/or condition (e.g., hemorrhage) which reduces the severity of the disease, disorder, and/or conditions or slows the progression of the disease, disorder, or condition (“therapeutic treatment”), and which can inhibit the disease, disorder, and/or condition (e.g., hemorrhage).

As used herein, and unless otherwise specified, a “therapeutically effective amount” of is an amount sufficient to provide a therapeutic benefit in the treatment of the disease, disorder and/or condition (e.g., hemorrhage) or to delay or minimize one or more symptoms associated with the disease, disorder, and/or condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder, and/or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease, disorder and/or condition, or enhances the therapeutic efficacy of another therapeutic agent.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The present disclosure is controlling to the extent it conflicts with any incorporated publication.

As used herein and in the appended claims, the term “platelet” can include whole platelets, fragmented platelets, platelet derivatives, or thrombosomes. “Platelets” within the above definition may include, for example, platelets in whole blood, platelets in plasma, platelets in buffer optionally supplemented with select plasma proteins, cold stored platelets, dried platelets, cryopreserved platelets, thawed cryopreserved platelets, rehydrated dried platelets, rehydrated cryopreserved platelets, lyopreserved platelets, thawed lyopreserved platelets, or rehydrated lyopreserved platelets. “Platelets” may be “platelets” of mammals, such as of humans, or such as of non-human mammals.

Thus, for example, reference to “anti-fibrinolytic loaded platelets” may be inclusive of anti-fibrinolytic loaded platelets as well as anti-fibrinolytic loaded platelet derivatives or anti-fibrinolytic loaded thrombosomes, unless the context clearly dictates a particular form.

As used herein, “thrombosomes” (sometimes also herein called “Tsomes” or “Ts”, particularly in the Examples and Figures) are platelet derivatives that have been treated with an incubating agent (e.g., any of the incubating agents described herein) and lyopreserved (e.g., freeze-dried to form thrombosomes). In some cases, thrombosomes can be prepared from pooled platelets. Thrombosomes can have a shelf life of 2-3 years in dry form at ambient temperature and can be rehydrated with sterile water within minutes for immediate infusion.

As used herein and in the appended claims, the term “fresh platelet” includes platelets stored for less than approximately 24 hours.

As used herein and in the appended claims the term “stored platelet” includes platelets stored for approximately 24 hours or longer before use.

As used herein and in the appended claims the term “fixed platelet” includes platelets fixed with a formalin solution.

As used herein and in the appended claims the term “unloaded” includes platelets, platelet derivatives, and/or thrombosomes that are not loaded with an active agent, such as platelets, platelet derivatives, and/or thrombosomes that are not loaded with an anti-fibrinolytic.

In some embodiments, rehydrating the anti-fibrinolytic loaded platelets includes adding to the platelets an aqueous liquid. In some embodiments, the aqueous liquid is water. In some embodiments, the aqueous liquid is an aqueous solution. In some embodiments, the aqueous liquid is a saline solution. In some embodiments, the aqueous liquid is a suspension.

In some embodiments, the rehydrated platelets have coagulation factor levels showing all individual factors (e.g., Factors VII, VIII and IX) associated with blood clotting at 40 international units (IU) or greater.

As used herein, “coagulopathy” is a bleeding disorder in which the blood's ability to coagulate (e.g., form clots) is impaired. This condition can cause a tendency toward prolonged or excessive bleed (e.g., diathesis). In some embodiments, a coagulopathy is caused by a disease (e.g., congenital hemophilia). In some embodiments, a coagulopathy is a drug induced coagulopathy. In some embodiments, a coagulopathy is induced by an antiplatelet agent-induced coagulopathy. In some embodiments, a coagulopathy is induced by an anti-platelet agent.

Accordingly, also provided herein are methods and compositions that are suitable for treating drug-induced coagulopathy. Anticoagulant drugs, such as warfarin, heparin, and the novel oral anticoagulants (NOACs) class inhibit various plasma factors of the coagulation cascade, resulting in increased bleeding potential.

Anticoagulant drugs are common in the U.S. adult population and employ a wide variety of mechanisms to disable segments of the clotting cascade. Anticoagulants are used to treat a number of cardiac or thromboembolic events. For example, warfarin (e.g., COUMADIN®) is approved for the prophylaxis and treatment of venous thrombosis and its extension, pulmonary embolism; the prophylaxis and treatment of thromboembolic complications associated with atrial fibrillation and/or cardiac valve replacement; the reduction in the risk of death, recurrent myocardial infarction, and thromboembolic events such as stroke or systemic embolization after myocardial infarction (see, e.g., Prescribing Information for warfarin (COUMADIN®)). As another example, heparin is approved for the treatment of thrombophlebitis, phlebothrombosis, and cerebral, coronary, and retinal vessel thrombosis to prevent extension of clots and thromboembolic phenomena. It is also used prophylactically to prevent the occurrence of thromboembolism, and to prevent clotting during dialysis and surgical procedures, particularly vascular surgery. Other drugs that have anticoagulant properties can include agents that inhibit factor IIa (thrombin) (also called anti-IIa agents, thrombin inhibitors, or direct thrombin inhibitors, depending on the mechanism of action), including dabigatran (e.g., PRADAXA®), argatroban, and hirudin; and agents that inhibit factor Xa, including rivaroxaban (e.g., XARELTO®), apixaban (e.g., ELIQUIS®), edoxaban (e.g., SAVAYSA®), and fondaparinux (e.g., ARIXTRA®). Traditional anticoagulants can include warfarin (e.g., COUMADIN®) and heparin/LMWH (low molecular weight heparins). Additional anticoagulants include heparainoids, factor IX inhibitors, Factor XI inhibitors, Factor VIIa inhibitors, and Tissue Factor inhibitors.

As used herein, an “anticoagulant” is an antithrombotic that does not include antiplatelet agents. Examples of antiplatelet agents include aspirin, cangrelor, ticagrelor, clopidogrel (e.g., PLAVIX®), prasugrel eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), and abciximab (e.g., REOPRO®). Typically, agents that inhibit P2Y receptors (e.g., P2Y12), glycoprotein IIb/IIIa, or that antagonize thromboxane synthase or thromboxane receptors, are considered to be antiplatelet agents. Other mechanisms of antiplatelet agents are known. As used herein, aspirin is considered to be an antiplatelet agent but not an anticoagulant.

Agents that inhibit Factor IIa, VIIa, IX, Xa, XI, Tissue Factor, or vitamin K-dependent synthesis of clotting factors (e.g., Factor II, VII, IX, or X) or that activate antithrombin (e.g., antithrombin III) are anticoagulants for the purpose of the present disclosure.

Other mechanisms of anticoagulants are known. Non-limiting examples of anticoagulants include dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, and low molecular weight heparins (e.g., dalteparin, enoxaparin, tinzaparin, ardeparin, nadroparin, reveparin, danaparoid). Additional non-limiting examples of anticoagulants include tifacogin, Factor VIIai, SB249417, pegnivacogin (with or without anivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon, acenocoumarol, indandiones, and fluindione. In some embodiments, the anticoagulant is selected from the group consisting of dabigatran, argatroban, hirudin, rivaroxaban, apixaban, edoxaban, fondaparinux, warfarin, heparin, low molecular weight heparins, tifacogin, Factor VIIai, SB249417, pegnivacogin (with or without anivamersen), TTP889, idraparinux, idrabiotaparinux, SR23781A, apixaban, betrixaban, lepirudin, bivalirudin, ximelagatran, phenprocoumon, acenocoumarol, indandiones, and fluindione.

Overcoming the effect of an anticoagulant varies according to the anticoagulant drug pharmacological action. In the case of advanced notice, as in a pre-planned surgery, the anti-coagulant dose can sometimes be tailored back before the surgery; however, there may be cases where such a reduction in dose is not advisable. In the case where an anti-coagulant need reversing quickly (e.g., for emergency surgery), reversal agents are typically slow acting, expensive, or carry significant risk to the patient.

Warfarin (e.g., COUMADIN®)—Warfarin works to prevent the activity of vitamin K in the liver which is a necessary co-factor to produce multiple coagulation factors. Warfarin reversal can sometimes be done be by dosing vitamin K or prothrombin complex concentrate (PCC). Vitamin K is low-cost and slow acting (more than 24 hrs PO) but can pose significant risk of inducing thrombosis in the patient, while PCC is expensive at roughly $5000/dose.

Dabigatran (e.g., PRADAXA®)—Dabigatran is a direct inhibitor of thrombin. The monoclonal antibody therapy idarucizumab (e.g., PRAXBIND®, Boehringer-Ingelheim, Germany) at dose of 5 grams (at two dose intervals each 2.5 grams) can typically reverse the effects of dabigatran within a few minutes. One wholesale price is $3482.50 for such a treatment.

Rivaroxaban (e.g., XARELTO®)—Rivaroxaban is a direct Factor Xa inhibitor. Rivaroxaban is reversed by Andexanet Alfa (e.g., ANDEXXA®), a recombinant Factor Xa decoy. This treatment can cost roughly $50,000 for a high-dose treatment.

Apixaban (e.g., ELIQUIS®)—Apixaban is a direct Factor Xa inhibitor. Apixaban is reversed by Andexanet Alfa, a recombinant Factor Xa decoy. This treatment costs roughly can cost $50,000 for a high-dose treatment.

Edoxaban (e.g., SAVAYSA®, LIXIANA®)—Edoxaban is a direct Factor Xa inhibitor. Exoxaban does not have an approved reversal agent. Ciraparantag (aripazine) and Andexanet Alfa have not been clinically proven to be appropriate.

Heparin and low molecular weight heparins are activators of antithrombin III (AT). AT inactivates proteases such as thrombin and Factor Xa. Protamine sulfate is a highly positively-charged polypeptide that binds to the negatively charged heparin and prevents its action on AT. Protamine sulfate is typically dosed at about 1.0 to about 1.5 mg/100 IU of active heparin.

Platelet-derived products (e.g., thrombosomes, cryo-preserved platelets are not currently used as a treatment method for anticoagulant drugs. In some embodiments, platelet derived products, including anti-fibrinolytic loaded platelets and anti-fibrinolytic loaded thrombosomes (e.g., freeze-dried platelets) are used to as a treatment method for anticoagulant drugs.

Treatments for anticoagulant drugs are not necessarily targeted antidotes. Some novel anticoagulant treatments, such as Andexanet Alfa (e.g., ANDEXXA®), have seen some success, yet can be expensive. As such, emergency treatments (pre-op, trauma, and the like) are typically blanket precautions to avoid or mitigate hemorrhage. Non-limiting examples include infusion of plasma, red blood cells, and anti-fibrinolytics. Products and methods are described herein for controlling bleeding and improving healing. The products and methods described herein can also be used to counteract the activity of an anticoagulant.

Products and methods are described herein for controlling bleeding and improving healing. The products and methods described herein can also be used to counteract the activity of an anticoagulant (e.g., warfarin (e.g., COUMADIN®), heparin, LMWH, dabigatran (e.g., PRADAXA®), argatroban, hirudin, rivaroxaban (e.g., XARELTO®), apixaban (e.g., ELIQUIS®), edoxaban (e.g., SAVAYSA®), fondaparinux (e.g., ARIXTRA®). The products and methods described herein are directed toward embodiments that aid in the closure and healing of wounds.

In certain embodiments, a composition comprising platelets such as lyophilized platelets or platelet derivatives may be delivered to a wound on the surface of or in the interior of a patient. In various embodiments, a composition comprising platelets or platelet derivatives can be applied in selected forms including, but not limited to, adhesive bandages, compression bandages, liquid solutions, aerosols, matrix compositions, and coated sutures or other medical closures. In some embodiments, a platelet derivative may be administered to all or only a portion of an affected area on the surface of a patient. In other embodiments, a composition comprising platelets such as lyophilized platelets or platelet derivatives may be administered systemically, for example via the blood stream. In embodiments, an application of the anti-fibrinolytic loaded platelet derivative can produce hemostatic effects for 2 or 3 days, preferably 5 to 10 days, or most preferably for up to 14 days.

As used herein, an “antiplatelet agent” is an antithrombotic and does not include anticoagulants. Examples of antiplatelet agents include aspirin (also called acetylsalicylic acid or ASA), cangrelor (e.g., KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), prasugrel (e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), and abciximab (e.g., REOPRO®). For the purpose of this disclosure, antiplatelet agents include agents that inhibit P2Y receptors (e.g., P2Y12), glycoprotein IIb/IIIa, or that antagonize thromboxane synthase or thromboxane receptors. Non-limiting examples of thromboxane A2 antagonists are aspirin, terutroban, and picotamide. Non-limiting examples of P2Y receptor antagonists include cangrelor, ticagrelor, elinogrel, clopidogrel, prasugrel, and ticlopidine. Non-limiting examples of glycoprotein IIb/IIIa include abciximab, eptifibatide, and tirofiban. NSAIDS (e.g., ibuprofen) are also considered to be antiplatelet agents for the purposes of this disclosure. Other mechanisms of anti-platelet agents are known. Antiplatelet agents also include PAR1 antagonists, PAR4 antagonists GPVI antagonists and alpha2beta1 collagen receptor antagonists. Non-limiting examples of PAR-1 antagonists include vorapaxar and atopaxar. As used herein, aspirin is considered to be an antiplatelet agent but not an anticoagulant. Additional non-limiting examples of antiplatelet agents include cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, and sarpogrelate.

In some embodiments, an antiplatelet agent can be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, and combinations thereof. In some embodiments, an antiplatelet agent can be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, and combinations thereof. In some embodiments, an antiplatelet agent can be selected from the group consisting of aspirin, cangrelor, ticagrelor, clopidogrel, prasugrel, eptifibatide, tirofiban, abciximab, terutroban, picotamide, elinogrel, ticlopidine, ibuprofen, vorapaxar, atopaxar, cilostazol, prostaglandin E1, epoprostenol, dipyridamole, treprostinil sodium, sarpogrelate and combinations thereof. In some embodiments, the antiplatelet agent can include multiple antiplatelet agents, such as 2 (or more) of any of the antiplatelet agents described herein. In some embodiments, the antiplatelet agent can be aspirin and clopidogrel.

Cangrelor like clopidogrel, ticagrelor, and prasugrel, blocks the P2Y12 (ADP) receptor on platelets. Cangrelor can in some cases be used as a representative of this class of drug. Cangrelor, unlike clopidogrel and prasugrel, does not need hepatic metabolism to become biologically active.

Eptifibatide is a peptide therapeutic that blocks the fibrin binding role of GPIIb-IIIa receptor on platelets. The drug is typically administered via IV as a 180 μg/kg bolus followed by 2 μg/kg/min continuous infusion. The blood concentration of eptifibatide is typically about 1-2 μM. Bleeding times generally return to normal within about 1 hour of drug stoppage.

Aspirin is an irreversible cylcooxygenase (COX) inhibitor. The COX enzyme in platelets is responsible for synthesis of thromboxane A2, prostaglandin E2 and prostacyclin (PGI2). Aspirin permanently inactivates the COX enzyme within platelets, and since platelets do not have the nuclear material to synthesize new enzyme, new platelets must be produced to overcome the aspirin effect. Without thromboxane A2, prostaglandin E2, and prostacyclin (PGI2) platelets are limited in their pro-aggregation activity. Many people are maintained on a low dose of aspirin to prevent unwanted clotting events. Aspirin bioavailability largely varies with administration route, with a single 500 mg dose IV at peaks of 500 μM and the same dose orally at 44 μM.

The antiplatelet class of drugs is widely used to prevent unwanted clotting episodes that lead to heart failure, stroke, and the like. In many cases, an antiplatelet drug may need to be reversed or stopped. In the case of advanced notice, as in a pre-planned surgery situation, the antiplatelet drug dose can sometimes be stopped before the surgery, preventing unwanted bleeding during surgery. In the case where an antiplatelet agent needs reversing quickly, reversal agents are typically not readily available, are expensive, or carry significant risk to the patient. In the case of need for rapid antiplatelet reversal, a platelet transfusion is typically administered, though the response to this is often only partial reversal. The caveat of this course of reversal is that the newly-infused platelets themselves are susceptible to circulating drug antiplatelet activity whereas, in some embodiments, compositions as described herein (e.g., including thrombosomes) are not. In some embodiments, compositions as described herein (e.g., including thrombosomes) are an active reversal agent. In some embodiments, the hemostatic activity of compositions as described herein (e.g., including thrombosomes) does not succumb to antiplatelet drugs.

Some exemplary antiplatelet agents and potential methods of reversal are described below.

Acetylsalicylic acid (ASA; aspirin)—aspirin acts as a COX-1 blocker in platelets, which renders the platelet inactive by irreversibly inhibiting platelet-derived thromboxane formation. Clinically, aspirin is sometimes reversed by a platelet transfusion in emergency situations or by stopping treatment where surgery is scheduled in the future.

Clopidogrel (e.g., PLAVIX®)—clopidogrel acts as to prevent ADP from binding to its receptor on platelets. ADP binding leads to platelet shape change and aggregation. Clopidogrel is non-reversible. Clinically, clopidogrel is sometimes reversed by a platelet transfusion in emergency situations or by stopping treatment where surgery is scheduled in the future.

Cangrelor (e.g., KENGREAL®)—cangrelor acts to prevent ADP from binding to its receptor on platelets. ADP binding leads to platelet shape change and aggregation. Clopidogrel is reversible and platelet function is returned approximately 1 hour after stopping infusion. Clinically it is generally preferred when reversal is needed after a procedure.

Ticagrelor (e.g., BRILINTA®)—ticagrelor acts to prevent ADP from binding to its receptor and acts as an inverse agonist. Ticagrelor is reversible and platelet function can return after approximately 72 hours of the last dosage. Reversal of action of ticagrelor can be affected by the time after the last dose. If the last dose was longer than 24 hours previous, then platelet transfusion can sometimes be therapeutic to reverse the results.

Effient (e.g., PRASUGREL®)—Effient acts to prevent ADP from binding to its receptor and acts as a non-reversable antagonist. It being a non-reversible antagonist, new platelets must be formed to overcoming its effect. Clinically Effient is reversed by a platelet transfusion in emergency situations or by stopping treatment where surgery is scheduled in the future.

Eptifibatide (Integrilin) —Eptifibatide acts to block the GpIIb/IIIa and acts as a reversible antagonist. Clinically, Integrilin is reversed by a platelet transfusion in emergency situations or by stopping treatment where surgery is scheduled in the future.

Platelet-derived products are not currently used as a treatment method for anticoagulant/antiplatelet drugs, and there are no currently approved reversal agents for antiplatelet agents. As such, emergency treatments (pre-op, trauma, and the like) are typically blanket precautions to avoid or mitigate hemorrhage. Non-limiting examples include infusion of plasma, red blood cells, and anti-fibrinolytics. Platelet derivatives (e.g., lyopreserved platelets (thrombosomes)) may be an effective alternative or supplement to these general treatments.

Products and methods are described herein for controlling bleeding and improving healing. The products and methods described herein can also be used to counteract the activity of an antiplatelet agent (e.g., aspirin (also called acetylsalicylic acid or ASA), cangrelor (e.g., KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), prasugrel (e.g., EFFIENT®), eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), or abciximab (e.g., REOPRO®)). The products and methods described herein are directed toward embodiments that can aid in the closure and healing of wounds.

Unless specified as loaded, platelets (e.g., freeze-dried (e.g., lyophilized) platelets), or platelet derivatives may or may not have not been loaded with a therapeutic agent (e.g., an anti-fibrinolytic). Thus, for example, the following: in some embodiments, platelets (e.g., anti-fibrinolytic loaded platelets), lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) are used to treat a coagulopathy contemplates both the use of unloaded platelets, unloaded lyophilized platelets, or unloaded platelet derivatives and the use of anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded lyophilized platelets, or anti-fibrinolytic loaded platelet derivatives, or a combination thereof to treat a coagulopathy.

In some embodiments, the dried platelets, such as freeze-dried platelets, have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% crosslinking of platelet membranes via proteins and/or lipids present on the membranes. In some embodiments, the dried platelets, such as freeze dried platelets, have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% crosslinking of platelet membranes via proteins and/or lipids present on the membranes. In some embodiments, the rehydrated platelets, have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% crosslinking of platelet membranes via proteins and/or lipids present on the membranes. In some embodiments, the rehydrated platelets, have between about 0.01% to about 5%, such as between about 0.1% to about 4%, such as between about 1% to between about 3%, such as between about 1% to about 2%, crosslinking of platelet membranes via proteins and/or lipids present on the membranes. In some embodiments, the rehydrated platelets, have at least about 1% to at least about 10, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% crosslinking of platelet membranes via proteins and/or lipids present on the membranes.

In some embodiments, the anti-fibrinolytic loaded platelets and the dried platelets, such as freeze-dried platelets, having a particle size (e.g., diameter, max dimension) of at least about 0.2 μm (e.g., at least about 0.3 μm, at least about 0.4 μm, at least about 0.5 μm, at least about 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at least about 0.9 μm, at least about 1.0 μm, at least about 1.0 μm, at least about 1.5 μm, at least about 2.0 μm, at least about 2.5 μm, or at least about 5.0 μm). In some embodiments, the particle size is less than about 5.0 μm (e.g., less than about 2.5 μm, less than about 2.0 μm, less than about 1.5 μm, less than about 1.0 μm, less than about 0.9 μm, less than about 0.8 μm, less than about 0.7 μm, less than about 0.6 μm, less than about 0.5 μm, less than about 0.4 μm, or less than about 0.3 μm). In some embodiments, the particle size is from about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

In some embodiments, at least 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of platelets and/or the dried platelets, such as freeze-dried platelets, have a particle size in the range of about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm). In some embodiments, at most 99% (e.g., at most about 95%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, or at most about 50%) of platelets and/or the dried platelets, such as freeze-dried platelets, are in the range of about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm). In some embodiments, about 50% to about 99% (e.g., about 55% to about 95%, about 60% to about 90%, about 65% to about 85, about 70% to about 80%) of platelets and/or the dried platelets, such as freeze-dried platelets, are in the range of about 0.3 μm to about 5.0 μm (e.g., from about 0.4 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

In some embodiments, (e.g., using unloaded platelets or platelet derivatives), the platelets or platelet derivatives are prepared consistent with the procedures described in U.S. Pat. No. 8,486,617 (such as, e.g., Examples 1-5) and U.S. Pat. No. 8,097,403 (such as, e.g., Examples 1-3).

Also provided herein are methods of preparing anti-fibrinolytic loaded platelets. In some embodiments, platelets are isolated prior to contacting the platelets with an anti-fibrinolytic.

Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; and step (b) contacting the platelets with an anti-fibrinolytic, and with a loading buffer comprising a salt, a base, a loading agent, and optionally ethanol, to form the anti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; step (b) contacting the platelets with an anti-fibrinolytic to form a first composition; and step (c) contacting the first composition with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form the anti-fibrinolytic loaded platelets.

In some embodiments, suitable organic solvents include, but are not limited to alcohols, esters, ketones, ethers, halogenated solvents, hydrocarbons, nitriles, glycols, alkyl nitrates, water or mixtures thereof. In some embodiments, suitable organic solvents includes, but are not limited to methanol, ethanol, n-propanol, isopropanol, acetic acid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropyl ether (IPE), tert-butyl methyl ether, dioxane (e.g., 1,4-dioxane), acetonitrile, propionitrile, methylene chloride, chloroform, toluene, anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane, dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone, dimethylacetamide, and combinations thereof.

Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; step (b) contacting the platelets with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and step (c) contacting the first composition with an anti-fibrinolytic, to form the anti-fibrinolytic loaded platelets.

In some embodiments, isolating platelets includes isolating platelets from blood.

In some embodiments, platelets are donor-derived platelets. In some embodiments, platelets are obtained by a process that includes an apheresis step. In some embodiments, platelets are fresh platelets. In some embodiments, platelets are stored platelets.

In some embodiments, platelets are derived in vitro. In some embodiments, platelets are derived or prepared in a culture prior to contacting the platelets with an anti-fibrinolytic. In some embodiments, preparing the platelets includes deriving or growing the platelets from a culture of megakaryocytes. In some embodiments, preparing the platelets includes deriving or growing the platelets (or megakaryocytes) from a culture of human pluripotent stem cells (PCSs), including embryonic stem cells (ESCs) and/or induced pluripotent stem cells (iPSCs).

Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets includes: step (a) preparing platelets; and step (b) contacting the platelets with an anti-fibrinolytic and with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets includes: step (a) preparing platelets; step (b) contacting the platelets with an anti-fibrinolytic to form a first composition; and step (c) contacting the first composition with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets includes: step (a) preparing platelets; step (b) contacting the platelets with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and step (c) contacting the first composition with an anti-fibrinolytic, to form the anti-fibrinolytic loaded platelets.

In some embodiments, no solvent is used. Thus, in some embodiments, the method for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) isolating platelets, for example in a liquid medium; and     -   b) contacting the platelets with an anti-fibrinolytic and with a         loading buffer comprising a salt, a base, and a loading agent,         to form the anti-fibrinolytic loaded platelets,         -   wherein the method does not comprise contacting the             platelets with an organic solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) isolating platelets, for example in a liquid medium;     -   b) contacting the platelets with an anti-fibrinolytic to form a         first composition; and     -   c) contacting the first composition with a buffer comprising a         salt, a base, and a loading agent, to form the anti-fibrinolytic         loaded platelets,         -   wherein the method does not comprise contacting the             platelets with an organic solvent such as ethanol and the             method does not comprise contacting the first composition             with an organic solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) isolating platelets, for example in a liquid medium;     -   b) contacting the platelets with a buffer comprising a salt, a         base, and a loading agent, to form a first composition; and     -   c) contacting the first composition with an anti-fibrinolytic,         to form the anti-fibrinolytic loaded platelets.         -   wherein the method does not comprise contacting the             platelets with an organic solvent such as ethanol and the             method does not comprise contacting the first composition             with an organic solvent such as ethanol.

In some embodiments, the method for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) preparing platelets;         -   and     -   b) contacting the platelets with an anti-fibrinolytic and with a         loading buffer comprising a salt, a base, and a loading agent,         to form the anti-fibrinolytic loaded platelets,         -   wherein the method does not comprise contacting the             platelets with an organic solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) preparing platelets;     -   b) contacting the platelets with an anti-fibrinolytic to form a         first composition; and     -   c) contacting the first composition with a buffer comprising a         salt, a base, and a loading agent, to form the anti-fibrinolytic         loaded platelets,     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol and the method does not         comprise contacting the first composition with an organic         solvent such as ethanol.

Thus, in some embodiments, the method for preparing anti-fibrinolytic loaded platelets comprises:

-   -   a) preparing platelets;     -   b) contacting the platelets with a buffer comprising a salt, a         base, and a loading agent, to form a first composition; and     -   c) contacting the first composition with an anti-fibrinolytic,         to form the anti-fibrinolytic loaded platelets.     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol and the method does not         comprise contacting the first composition with an organic         solvent such as ethanol.

In some embodiments, the loading agent is a saccharide. In some embodiments, the saccharide is a monosaccharide. In some embodiments, the saccharide is a disaccharide. In some embodiments, the saccharide is a non-reducing disaccharide. In some embodiments, the saccharide is sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the loading agent is a starch. In some embodiments, a loading agent is a cryoprotectant. In some embodiments, (e.g., for platelets or platelet derivatives not loaded with an anti-fibrinolytic agent), a “loading agent” can be used in the preparation of the platelets or platelet derivatives, for example, as part of an incubating agent.

As used herein, the term “anti-fibrinolytic,” “anti-fibrinolytics,” or “anti-fibrinolytic compound,” is any compound capable of inhibiting fibrinolysis. Fibrinolysis is the process where the activated plasminogen removes excess fibrin and promotes fibrin clot formation and wound healing (Szekely, A. and Lex, D. J., Antifibrinolytics, Heart Lung Vessel, 6(1): 5-7, (2014), which is incorporated herein by reference in its entirety). Inhibiting fibrinolysis can be useful under certain conditions. For example, in the case of traumatic bleeding events and/or hemorrhage, inhibiting fibrinolysis can enhance the formation of blood clots (e.g., stopping bleeding).

In some embodiments, the anti-fibrinolytic can be ε-aminocaproic acid. In some embodiments, the anti-fibrinolytic can be tranexamic acid. In some embodiments, the anti-fibrinolytic can be aprotinin. In some embodiments, the anti-fibrinolytic can be aminomethylbenzoic acid. In some embodiments, the anti-fibrinolytic can be fibrinogen. In some embodiments, the anti-fibrinolytic can be a combination of two or more anti-fibrinolytics.

In some embodiments, an anti-fibrinolytic (e.g., EACA) loaded into platelets is modified to include an imaging agent. For example, an anti-fibrinolytic can be modified with an imaging agent in order to image the anti-fibrinolytic loaded platelet in vivo. In some embodiments, an anti-fibrinolytic can be modified with two or more imaging agents (e.g., any two or more of the imaging agents described herein). In some embodiments, an anti-fibrinolytic loaded into platelets is modified with a radioactive metal ion, a paramagnetic metal ion, a gamma-emitting radioactive halogen, a positron-emitting radioactive non-metal, a hyperpolarized NMR-active nucleus, a reporter suitable for in vivo optical imaging, or a beta-emitter suitable for intravascular detection. For example, a radioactive metal ion can include, but is not limited to, positron emitters such as ⁵⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ⁹⁴Tc or ⁶⁸Ga; or gamma-emitters such as ¹⁷¹Tc, ¹¹¹In, ¹³In, or ⁶⁷Ga. For example, a paramagnetic metal ion can include, but is not limited to Gd(III), a Mn(II), a Cu(II), a Cr(III), a Fe(III), a Co(II), a Er(II), a Ni(II), a Eu(III) or a Dy(III), an element comprising an Fe element, a neodymium iron oxide (NdFeO₃) or a dysprosium iron oxide (DyFeO₃). For example, a paramagnetic metal ion can be chelated to a polypeptide or a monocrystalline nanoparticle. For example, a gamma-emitting radioactive halogen can include, but is not limited to ¹²³I, ¹³¹I or ⁷⁷Br. For example, a positron-emitting radioactive non-metal can include, but is not limited to ¹¹C, ¹³N, ¹⁵O, ¹⁷F, ¹⁸F, ⁷⁵Br, ⁷⁶Br or 1241 For example, a hyperpolarized NMR-active nucleus can include, but is not limited to ¹³C, ¹⁵N, ¹⁹F, ²⁹Si and ³¹P. For example, a reporter suitable for in vivo optical imaging can include, but is not limited to any moiety capable of detection either directly or indirectly in an optical imaging procedure. For example, the reporter suitable for in vivo optical imaging can be a light scatterer (e.g., a colored or uncolored particle), a light absorber or a light emitter. For example, the reporter can be any reporter that interacts with light in the electromagnetic spectrum with wavelengths from the ultraviolet to the near infrared. For example, organic chromophoric and fluorophoric reporters include groups having an extensive delocalized electron system, e.g. cyanines, merocyanines, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, benzothiaphenothiazinium dyes, anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecular charge-transfer dyes and dye complexes, tropones, tetrazines, b/s(dithiolene) complexes, bis(benzene-dithiolate) complexes, iodoaniline dyes, b/stS.O-dithiolene) complexes. For example, the reporter can be, but is not limited to a fluorescent, a bioluminescent, or chemiluminescent polypeptide. For example, a fluorescent or chemiluminescent polypeptide is a green florescent protein (GFP), a modified GFP to have different absorption/emission properties, a luciferase, an aequorin, an obelin, a mnemiopsin, a berovin, or a phenanthridinium ester. For example, a reporter can be, but is not limited to rare earth metals (e.g., europium, samarium, terbium, or dysprosium), or fluorescent nanocrystals (e.g., quantum dots). For example, a reporter may be a chromophore that can include, but is not limited to fluorescein, sulforhodamine 101 (Texas Red), rhodamine B, rhodamine 6G, rhodamine 19, indocyanine green, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green 514, tetramethylrhodamine, and Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. For example, a beta-emitter can include, but is not limited to radio metals ⁶⁷Cu, ⁸⁹Sr, ⁹⁰Y, ¹⁵³Sm, ¹⁸⁵Re, ¹⁸⁸Re or ¹⁹²Ir, and non-metals ³²P, ³³P, ³¹S, ³⁸Cl, ³⁹Cl, ⁸²Br and ⁸³Br. In some embodiments, an anti-fibrinolytic loaded into platelets can be associated with gold or other equivalent metal particles (such as nanoparticles). For example, a metal particle system can include, but is not limited to gold nanoparticles (e.g., Nanogold™).

In some embodiments, an anti-fibrinolytic loaded into platelets that is modified with an imaging agent is imaged using an imaging unit. The imaging unit can be configured to image the anti-fibrinolytic loaded platelets in vivo based on an expected property (e.g., optical property from the imaging agent) to be characterized. For example, imaging techniques (in vivo imaging using an imaging unit) that can be used, but are not limited to are: computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI). Chen, Z., et al., Advance of Molecular Imaging Technology and Targeted Imaging Agent in Imaging and Therapy, Biomed Res Int., 819324, doi: 10.1155/2014/819324 (2014) have described various imaging techniques and which is incorporated by reference herein in its entirety.

For example, a modified anti-fibrinolytic can be modified such that the modifying group interacts with the anti-fibrinolytic. In a non-limiting way a modifying agent such as dansyl chloride can interact with the anti-fibrinolytic. Dansyl chloride can interact with primary amino groups in aliphatic and aromatic amines and can produce blue or blue-green sulfonamide adducts. In some embodiments, dansyl chloride can interact with EACA to generate a modified anti-fibrinolytic (e.g., dansyl-EACA).

In some embodiments, such as embodiments wherein the platelets are treated with the an anti-fibrinolytic (e.g., EACA) and the buffer sequentially as disclosed herein, the anti-fibrinolytic can be loaded in a liquid medium that can be modified to change the proportion of media components or to exchange components for similar products, or to add components necessary for a given application.

In some embodiments, the loading buffer and/or the liquid medium include one or more of a) water or a saline solution, b) one or more additional salts, or c) a base. In some embodiments, the loading buffer, and/or the liquid medium, may include one or more of a) DMSO, b) one or more salts, or c) a base.

In some embodiments, the loading agent is loaded into the platelets in the presence of an aqueous medium. In some embodiments, the loading agent is loaded in the presence of a medium comprising DMSO. As an example, one embodiment of the methods herein includes contacting platelets with an anti-fibrinolytic and with an aqueous loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets. As an example, one embodiment of the methods herein includes contacting platelets with an anti-fibrinolytic and with a loading buffer comprising DMSO and comprising a salt, a base, a loading agent, and optionally ethanol, to form the anti-fibrinolytic loaded platelets.

In some embodiments, the loading buffer and/or the liquid medium, include one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salt that can be found in blood or blood products, or that is known to be useful in drying platelets, or any combination of two or more of these.

Preferably, these salts are present in the composition at an amount that is about the same as is found in whole blood.

In some embodiments, the loading buffer and/or liquid medium further comprises a carrier protein. In some embodiments, the carrier protein comprises human serum albumin, bovine serum albumin, or a combination thereof. In some embodiments, the carrier protein is present in an amount of about 0.05% to about 1.0% (w/v).

In some embodiments, the anti-fibrinolytic loaded platelets are prepared by incubating the platelets with the anti-fibrinolytic in the liquid medium for different durations at or at different temperatures from about 15-45° C., or about 37° C. The step of incubating the platelets to load one or more anti-fibrinolytic compounds includes incubating the platelets for a time suitable for loading, as long as the time, taken in conjunction with the temperature, is sufficient for the anti-fibrinolytic to come into contact with the platelets and, preferably, be incorporated, at least to some extent, into the platelets. In some embodiments, the anti-fibrinolytic loaded platelets are prepared by incubating the platelets with the anti-fibrinolytic in the liquid medium at a temperature from about 18-42° C., about 20-40° C., about 22-37° C., or about 16° C., about 18° C., about 20° C., about 22° C., about 24° C., about 26° C., about 28° C., about 30° C., about 32° C., about 34° C., about 36° C., about 37° C., about 39° C., about 41° C., about 43° C., or about 45° C. for at least about 5 minutes (mins) (e.g., at least about 20 mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30 hrs, about 36 hrs, about 42 hrs, about 48 hrs, or at least about 48 hrs. In some embodiments, the anti-fibrinolytic loaded platelets are prepared by incubating the platelets with the anti-fibrinolytic in the liquid medium at a temperature from about 18-42° C., about 20-40° C., about 22-37° C., or about 16° C., about 18° C., about 20° C., about 22° C., about 24° C., about 26° C., about 28° C., about 30° C., about 32° C., about 34° C., about 36° C., about 37° C., about 39° C., about 41° C., about 43° C., or about 45° C. for no more than about 48 hrs (e.g., no more than about 20 mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30 hrs, about 36 hrs, or no more than about 42 hrs). In some embodiments, the anti-fibrinolytic loaded platelets are prepared by incubating the platelets with the anti-fibrinolytic in the liquid medium from about 10 mins to about 48 hours (e.g., from about 20 mins to about 36 hrs, from about 30 mins to about 24 hrs, from about 1 hr to about 20 hrs, from about 2 hrs to about 16 hours, from about 10 mins to about 24 hours, from about 20 mins to about 12 hours, from about 30 mins to about 10 hrs, or from about 1 hr to about 6 hrs.

In one embodiment, contacting platelets with an anti-fibrinolytic includes contacting the platelets with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent for a period of time, such as a period of 1 minute to 48 hours, such as 2 hours.

In some embodiments, the platelets are at a concentration from about 1,000 platelets/μl to about 10,000,000 platelets/μl. In some embodiments, the platelets are at a concentration from about 50,000 platelets/μl to about 4,000,000 platelets/μl. In some embodiments, the platelets are at a concentration from about 100,000 platelets/μl to about 300,000,000 platelets/μl. In some embodiments, the platelets are at a concentration from about 1,000,000 to about 2,000,000. In some embodiments, the platelets are at a concentration of about 200,000,000 platelets/μl.

In some embodiments of the methods of preparing anti-fibrinolytic loaded platelets disclosed herein, the methods further include acidifying the platelets, or pooled platelets, to a pH of about 6.0 to about 7.4, prior to a contacting step disclosed herein. In some embodiments, the methods include acidifying the platelets to a pH of about 6.5 to about 6.9. In some embodiments, the methods include acidifying the platelets to a pH of about 6.6 to about 6.8. In some embodiments, the acidifying includes adding to the pooled platelets a solution comprising Acid Citrate Dextrose.

In some embodiments, the platelets are isolated prior to a contacting step. In some embodiments, the methods further include isolating platelets by using centrifugation. In some embodiments, the centrifugation occurs at a relative centrifugal force (RCF) of about 800 g to about 2000 g. In some embodiments, the centrifugation occurs at relative centrifugal force (RCF) of about 1300 g to about 1800 g. In some embodiments, the centrifugation occurs at relative centrifugal force (RCF) of about 1500 g. In some embodiments, the centrifugation occurs for about 1 minute to about 60 minutes. In some embodiments, the centrifugation occurs for about 10 minutes to about 30 minutes. In some embodiments, the centrifugation occurs for about 20 minutes.

In some embodiments, the platelets are at a concentration from about 1,000 platelets/μl to about 10,000,000 platelets/l. In some embodiments, the platelets are at a concentration from about 50,000 platelets/μl to about 4,000,000 platelets/μl. In some embodiments, the platelets are at a concentration from about 100,000 platelets/μl to about 300,000,000 platelets/l. In some embodiments, the platelets are at a concentration from about 1,000,000 to about 2,000,000. In some embodiments, the platelets are at a concentration of about 2,000,000 platelets/l.

In some embodiments, the buffer is a loading buffer comprising the components as listed in Table 1 herein. In some embodiments, a loading buffer is an incubating agent. In some embodiments, the loading buffer includes one or more salts, such as phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salt that can be found in blood or blood products. Exemplary salts include sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof. In some embodiments, the loading buffer includes from about 0.5 mM to about 100 mM of the one or more salts. In some embodiments, the loading buffer includes from about 1 mM to about 100 mM (e.g., about 2 mM to about 90 mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 to about 85 mM) about of the one or more salts. In some embodiments, the loading buffer includes about 5 mM, about 75 mM, or about 80 mM of the one or more salts.

In some embodiments, the loading buffer includes one or more buffers, e.g., N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and/or sodium-bicarbonate (NaHCO₃). In some embodiments, the loading buffer includes from about 5 to about 100 mM of the one or more buffers. In some embodiments, the loading buffer includes from about 5 to about 50 mM (e.g., from about 5 mM to about 40 mM, from about 8 mM to about 30 mM, about 10 mM to about 25 mM) about of the one or more buffers. In some embodiments, the loading buffer includes about 10 mM, about 20 mM, about 25 mM, or about 30 mM of the one or more buffers.

In some embodiments, the loading buffer includes one or more saccharides, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. In some embodiments, the loading buffer includes from about 10 mM to about 1,000 mM of the one or more saccharides. In some embodiments, the loading buffer includes from about 50 to about 500 mM of the one or more saccharides. In embodiments, one or more saccharides is present in an amount of from 10 mM 10 to 500 mM. In some embodiments, one or more saccharides is present in an amount of from 50 mM to 200 mM. In embodiments, one or more saccharides is present in an amount from 100 mM to 150 mM.

In some embodiments, the anti-fibrinolytic includes one anti-fibrinolytic. In some embodiments, the anti-fibrinolytic includes two or more anti-fibrinolytics.

In some embodiments, the methods further include incubating the anti-fibrinolytic (e.g., EACA) in the presence of the loading buffer prior to the treatment step. In some embodiments, the methods further include incubating the loading buffer and a solution comprising the anti-fibrinolytic and water at about 37° C. using different incubation periods. In some embodiments, the solution includes a concentration of about 1 μM to about 100 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 10 μM to about 10 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 100 μM to about 100 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 200 μM to about 1 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 300 μM to about 900 μM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 400 μM to about 800 μM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 500 μM to about 700 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 600 μM. In some embodiments, the solution includes a concentration of about 0.1 mM to about 1.0 M of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 1.0 mM to about 900 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 10 mM to about 800 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 50 mM to about 700 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 100 mM to about 600 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 150 mM to about 500 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 200 mM to about 400 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 250 mM to about 300 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.2 mM to about 9 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.3 mM to about 8 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.4 mM to about 7 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.5 mM to about 6 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.6 mM to about 5 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.7 mM to about 4 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.8 mM to about 3 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 0.9 mM to about 2 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 1 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 10 mM to about 150 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 20 mM to about 125 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 30 mM to 100 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 40 mM to about 90 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 50 mM to 80 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 60 mM to 70 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 50 mM of the anti-fibrinolytic. In some embodiments, the solution includes a concentration of about 100 mM of the anti-fibrinolytic.

In some embodiments, the incubation of the anti-fibrinolytic in the presence of the loading buffer is performed from about 1 minute to about 4 hours. In some embodiments, the incubation is performed at an incubation period of from about 30 minutes to about 3 hours. In some embodiments, the incubation is performed at an incubation period of from about 1 hour to about 2 hours. In some embodiments, the incubation is performed at an incubation period of about 3 hours.

In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 1 μM to about 100 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 10 μM to about 100 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 100 μM to about 10 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 200 μM to about 1 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 300 μM to about 900 μM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 400 μm to about 800 μM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 500 μm to about 700 μM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is about 600 μM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 0.1 mM to about 100 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 1.0 mM to about 900 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 10 mM to about 800 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 50 mM to about 700 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 100 mM to about 600 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 150 mM to about 500 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 200 mM to about 400 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 250 mM to about 300 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 1 mM to 100 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 5 mM to about 95 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 10 mM to about 90 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 15 mM to about 85 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 20 mM to about 80 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 25 mM to about 75 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 30 mM to about 70 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 35 mM to about 65 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 40 mM to about 60 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 45 mM to about 55 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 10 mM to about 100 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 20 mM to 90 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 30 mM to about 80 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 40 mM to 70 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is from about 50 mM to 60 mM. In some embodiments, the concentration of anti-fibrinolytic in the anti-fibrinolytic loaded platelets is about 50 mM. In some embodiments, the therapeutically effective amount can be any of the concentrations described herein.

In some embodiments, the methods further include drying the anti-fibrinolytic loaded platelets. In some embodiments, the drying step includes freeze-drying the anti-fibrinolytic loaded platelets. In some embodiments, the methods further include rehydrating the anti-fibrinolytic loaded platelets obtained from the drying step.

In some embodiments, anti-fibrinolytic loaded platelets are prepared by using any of the variety of methods provided herein.

In some embodiments, rehydrated anti-fibrinolytic loaded platelets are prepared by any one method comprising rehydrating the anti-fibrinolytic loaded platelets provided herein.

The anti-fibrinolytic loaded platelets can be used, for example, in therapeutic applications as disclosed herein. As described herein, platelets can stop bleeding by aggregating at an injury site which can be further alleviated by anti-fibrinolytic loaded platelets. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat traumatic bleeding events, such as a hemorrhage. Hemorrhage occurs when blood escapes outside its containing vessel (e.g., artery, vein, capillary, etc.) In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat an external hemorrhage. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat an internal hemorrhage. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat an external and an internal hemorrhage. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat a surgical hemorrhage. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat a non-surgical hemorrhage. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat a grade (e.g., category) 1 hemorrhage. For example, a grade 1 hemorrhage can include petechial bleeding. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat a grade 2 hemorrhage. For example, a grade (e.g., category) 2 hemorrhage can include mild blood loss (e.g., a clinically significant amount of blood). In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat a grade 3 hemorrhage. For example, a grade (e.g., category 3) hemorrhage can include gross blood loss and can require transfusion. In some embodiments, the anti-fibrinolytic loaded platelets can be used to treat a grade 4 hemorrhage. For example, a grade (e.g., category) 4 hemorrhage can include debilitating blood loss, retinal blood loss, and/or cerebral blood loss associated with a fatality.

Unloaded platelets can be used, for example, in therapeutic applications as disclosed herein. For example, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be used to treat a disease. In some embodiments, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be used to treat hemophilia. In some embodiments, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be used to treat classic hemophilia.

In some embodiments, treatment of a subject with platelets loaded with an anti-fibrinolytic compound provides a “r” time (time to clot) that is shorter than the “r” time for treatment of the subject with the same amount of the free anti-fibrinolytic compound, that is, the anti-fibrinolytic compound that is not loaded into the platelets.

In some embodiments, treatment of a subject with thrombosomes loaded with an anti-fibrinolytic compound provides a “r” time (time to clot) that is shorter than the “r” time for treatment of the subject with the same amount of the free anti-fibrinolytic compound, that is, the anti-fibrinolytic compound that is not loaded into the thrombosomes.

The anti-fibrinolytic loaded platelets can be used in therapeutic applications as disclosed herein. For example, the anti-fibrinolytic loaded platelets can be used to treat hemophilia. In some embodiments, the anti-fibrinolytics can be used to treat classic (e.g., inherited or congenital hemophilia). Hemophilia is a disease in which the ability of blood to clot is severely reduced. Hemophilia can cause a subject to severely bleed even from a slight injury. Classic hemophilia generally results from the deficiency of one or more clotting factors. The various types of hemophilia generally result from a deficiency in one more clotting factors, such as, in a non-limiting way, Factor VII, Factor VIII, and Factor IX. Other types of clotting factor deficiencies can also result in hemophilia, for example, clotting Factor XI. Some non-limiting examples of congenital hemophilia types include, for example, hemophilia A, hemophilia B, and hemophilia C.

Hemophilia A and hemophilia B diseases are congenital X-linked coagulation disorders caused by the lack of or a defect in the gene required to produce active Factor VIII or Factor IX protein, respectively. Hemophilia often manifests in patients experiencing frequent nosebleeds, easy bruising, and excessive bleeding during menstruation or invasive procedures. The disease affects about 20,000 people in the United States. There are also acquired forms of hemophilia that are caused by or exacerbated by neutralizing antibodies to Factor VIII or Factor IX. Factor VIII and Factor IX are produced in the liver. Plasma levels in a healthy human subject (“normal” levels) of Factor VIII are about 0.22 μg/mL and about 5 μg/mL for Factor IX. Factor VIII and Factor IX are coagulation factors of the intrinsic pathway to repair damaged surface of blood vessels. Additionally, thrombosomes potentiate clot formation and through alterative mechanisms reduce the time necessary to clot formation when the clotting process is reduced by the lack of Factor VIII or Factor IX.

In some embodiments, platelets (e.g., anti-fibrinolytic loaded platelets), lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) are used to treat a coagulopathy. In some embodiments, the coagulopathy is a drug-induced coagulopathy. In some embodiments, the coagulopathy occurs following administration of an antiplatelet agent. In some embodiments, the coagulopathy occurs following administration of an anticoagulant.

In some embodiments, a composition comprising platelets such as lyophilized platelets or platelet derivatives (any of which may be loaded to with anti-fibrinolytics) may be delivered to a wound on the surface of or in the interior of a patient. In some embodiments, a composition comprising platelets, lyophilized platelets, or platelet derivatives (any of which may be loaded to with anti-fibrinolytics) can be applied in selected forms including, but not limited to, adhesive bandages, compression bandages, liquid solutions, aerosols, matrix compositions, and coated sutures or other medical closures. In some embodiments, a platelet derivative (e.g. an anti-fibrinolytic loaded platelet derivative) may be administered to all or only a portion of an affected area on the surface of a patient. In other embodiments, a composition comprising platelets such lyophilized platelets or platelet derivatives (any of which may be loaded to with anti-fibrinolytics) may be administered systemically, for example via the blood stream. In some embodiments, an application of the platelet derivative (e.g. an anti-fibrinolytic loaded platelet derivative) can produce hemostatic effects for 2 or 3 days, preferably 5 to 10 days, or most preferably for up to 14 days.

Some embodiments provide a method of treating a coagulopathy in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets such as lyophilized platelets (e.g. anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g. anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of treating a coagulopathy in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some embodiments of any of the methods described herein, the coagulopathy is the result of an anticoagulant.

Some embodiments provide a method of treating coagulopathy in a subject, wherein the subject is a subject in need thereof, wherein the subject has been treated or is being treated with an anticoagulant, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of treating coagulopathy in a subject, wherein the subject has been treated or is being treated with an anticoagulant, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject is a subject in need thereof, wherein the subject has been treated or is being treated with an anticoagulant, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or anti-platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject is a subject in need thereof, wherein the subject has been treated or is being treated with an anticoagulant, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

Compositions as described herein can also be administered to prepare a subject for surgery, in some cases. For some patients taking an anticoagulant, it may be difficult or impossible to reduce the dosage of the anticoagulant before surgery (e.g., in the case of trauma or other emergency surgery). For some patients taking an anticoagulant, it may be inadvisable to reduce the dosage of the anticoagulant before surgery (e.g., if the patient would be at risk of a thrombotic event (e.g., deep vein thrombosis, pulmonary embolism, or stroke) if the dosage of the anticoagulant were reduced over time.

Accordingly, some embodiments provide a method of preparing a subject for surgery, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

Some embodiments provide a method of preparing a subject for surgery, wherein the subject has been treated or is being treated with an anticoagulant, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery, wherein the subject has been treated or is being treated with an anticoagulant, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some embodiments, a surgery can be an emergency surgery (e.g., in the case of trauma) or a scheduled surgery.

In some embodiments of any of the methods described herein, treatment with an anticoagulant can be stopped (e.g., in preparation for surgery). In some embodiments, treatment with an anticoagulant can continue.

In some embodiments of any of the methods described herein, the subject may or may not be also treated with an anticoagulant reversal agent (e.g., idarucizumab, Andexanet Alfa, Ciraparantag (aripazine), protamine sulfate, vitamin K). In some embodiments, the subject is not also treated with an anticoagulant reversal agent. In some embodiments, the subject is also treated with an anticoagulant reversal agent. It will be understood that an anticoagulant reversal agent can be chosen based on the anticoagulant administered to the subject.

Some embodiments provide a method of ameliorating the effects of an anticoagulant in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of ameliorating the effects of an anticoagulant in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some embodiments, the effects of an anticoagulant may need to be ameliorated due to an incorrect dosage of an anticoagulant. For example, in some embodiments, the effects of an anticoagulant can be ameliorated following an overdose of the anticoagulant. In some cases, the effects of an anticoagulant may need to be ameliorated due to a potential for interaction with another drug (e.g., a second anticoagulant). For example, in some embodiments, the effects of an anticoagulant can be ameliorated following an erroneous dosing of two or more drugs, at least one of which is an anticoagulant. In some cases, the composition is administered following administration to the subject or assumption by subject, or an overdose of the anticoagulant.

In some embodiments, the anticoagulant is selected from the group consisting of an anti-factor IIa agent such as dabigatran (e.g., PRADAXA®), argatroban, or hirudin; an anti-factor Xa agent such as rivaroxaban (e.g., XARELTO®), apixaban (e.g., ELIQUIS®), edoxaban (e.g., SAVAYSA®), or fondaparinux (e.g., ARIXTRA®); a traditional anticoagulant such as warfarin (e.g., COUMADIN®) and heparin/LMWH (low molecular weight heparins); supplements such as herbal supplements, and a combination thereof. Examples of supplements include garlic, coenzyme CoQ10, glucosamine, glucosamine-condroitin sulfate. A non-limiting example of an herbal supplement is garlic.

In some embodiments, the anticoagulant is dabigatran (e.g., PRADAXA®).

In some embodiments, the anticoagulant is argatroban.

In some embodiments, the anticoagulant is hirudin.

In some embodiments, the anticoagulant is rivaroxaban (e.g., XARELTO®).

In some embodiments, the anticoagulant is apixaban (e.g., ELIQUIS®).

In some embodiments, the anticoagulant is edoxaban (e.g., SAVAYSA®).

In some embodiments, the anticoagulant is fondaparinux (e.g., ARIXTRA®).

In some embodiments, the anticoagulant is heparin or a low molecular weight heparin (LMWH).

In some embodiments, the anticoagulant is warfarin (e.g., COUMADIN®).

In some embodiments, the anticoagulant is tifacogin.

In some embodiments, the anticoagulant is Factor VIIai.

In some embodiments, the anticoagulant is SB249417.

In some embodiments, the anticoagulant is pegnivacogin (with or without anivamersen).

In some embodiments, the anticoagulant is TTP889.

In some embodiments, the anticoagulant is idraparinux.

In some embodiments, the anticoagulant is idrabiotaparinux.

In some embodiments, the anticoagulant is SR23781A.

In some embodiments, the anticoagulant is apixaban.

In some embodiments, the anticoagulant is betrixaban.

In some embodiments, the anticoagulant is lepirudin.

In some embodiments, the anticoagulant is bivalirudin.

In some embodiments, the anticoagulant is ximelagatran.

In some embodiments, the anticoagulant is phenprocoumon.

In some embodiments, the anticoagulant is acenocoumarol.

In some embodiments, the anticoagulant an indandione.

In some embodiments, the anticoagulant is fluindione.

In some embodiments, the anticoagulant is a supplement.

In some embodiments, the anticoagulant is an herbal supplement.

In some embodiments, rehydrating the composition (e.g., any of the compositions described herein) comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) comprises adding to the platelets an aqueous liquid. In some embodiments, the aqueous liquid is water. In some embodiments, the aqueous liquid is an aqueous solution (e.g., a buffer). In some embodiments, the aqueous liquid is a saline solution. In some embodiments, the aqueous liquid is a suspension.

In some embodiments, the rehydrated platelets (e.g., anti-fibrinolytic loaded platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) have coagulation factor levels showing all individual factors (e.g., Factors VII, VIII and IX) associated with blood clotting at 40 international units (IU) or greater.

Some embodiments provide a method of treating coagulopathy in a subject, wherein the subject has been treated or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of platelets (e.g., anti-fibrinolytic loaded platelets), lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets), or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives).

Some embodiments provide a method of treating coagulopathy in a subject, wherein the subject has been treated or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject has or has been treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of restoring normal hemostasis in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject has been treated or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition. Some embodiments provide a method of restoring normal hemostasis in a subject, wherein the subject has been treated or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Compositions as described herein can also be administered to prepare a subject for surgery, in some cases. For some patients taking an antiplatelet agent, it may be difficult or impossible to reduce the dosage of the antiplatelet agent before surgery (e.g., in the case of trauma or other emergency surgery). For some patients taking an antiplatelet agent, it may be inadvisable to reduce the dosage of the antiplatelet agent before surgery (e.g., if the patient would be at risk of a thrombotic event (e.g., deep vein thrombosis, pulmonary embolism, or stroke) if the dosage of the antiplatelet agent were reduced over time.

Accordingly, some embodiments provide a method of preparing a subject for surgery, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

Some embodiments provide a method of preparing a subject for surgery, wherein the subject has been treated or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of preparing a subject for surgery, wherein the subject has been treated or is being treated with an antiplatelet agent, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some embodiments, a surgery can be an emergency surgery (e.g., in the case of trauma) or a scheduled surgery.

In some embodiments, treatment with an anticoagulant can be stopped (e.g., in preparation for surgery). In some embodiments, treatment with an anticoagulant can continue.

Some embodiments provide a method of ameliorating the effects of an antiplatelet agent in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) and contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.

Some embodiments provide a method of ameliorating the effects of an antiplatelet agent in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of a composition prepared by a process comprising contacting platelets (e.g., anti-fibrinolytic loaded platelets) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the composition.

In some cases, the effects of an antiplatelet agent may need to be ameliorated due to an incorrect dosage of an antiplatelet agent. For example, in some embodiments, the effects of an antiplatelet agent can be ameliorated following an overdose of the antiplatelet agent. In some cases, the effects of an antiplatelet agent may need to be ameliorated due to a potential for interaction with another drug (e.g., a second antiplatelet agent). For example, in some embodiments, the effects of an antiplatelet agent can be ameliorated following an erroneous dosing of two or more drugs, at least one of which is an antiplatelet agent. In some cases, the composition is administered following administration to the subject or assumption by subject, or an overdose of the antiplatelet agent.

In some embodiments, the antiplatelet agent is selected from the group consisting of aspirin (also called acetylsalicylic acid or ASA); a P2Y12 inhibitor such as cangrelor (e.g., KENGREAL®), ticagrelor (e.g., BRILINTA®), clopidogrel (e.g., PLAVIX®), or prasugrel (e.g., EFFIENT®); a glycoprotein IIb/IIIa inhibitor such as eptifibatide (e.g., INTEGRILIN®), tirofiban (e.g., AGGRASTAT®), or abciximab (e.g., REOPRO®)); supplements such as herbal supplements; or a combination of any thereof.

Examples of supplements include ginger, ginseng, ginkgo, green tea, kava, saw palmetto, boldo (Peumus boldus), Danshen (Salvia miltiorrhiza), Dong quai (Angelica sinensis) papaya (Carica papaya), fish oil, and vitamin E. Examples of herbal supplements include ginger, ginseng, and ginkgo.

In some embodiments, the antiplatelet agent is aspirin.

In some embodiments, the antiplatelet agent is cangrelor (e.g., KENGREAL®).

In some embodiments, the antiplatelet agent is ticagrelor (e.g., BRILINTA®).

In some embodiments, the antiplatelet agent is clopidogrel (e.g., PLAVIX®).

In some embodiments, the antiplatelet agent is prasugrel (e.g., EFFIENT®).

In some embodiments, the antiplatelet agent is eptifibatide (e.g., INTEGRILIN®).

In some embodiments, the antiplatelet agent is tirofiban (e.g., AGGRASTAT®).

In some embodiments, the antiplatelet agent is abciximab (e.g., REOPRO®).

In some embodiments, the antiplatelet agent is terutroban.

In some embodiments, the antiplatelet agent is picotamide.

In some embodiments, the antiplatelet agent is elinogrel.

In some embodiments, the antiplatelet agent is ticlopidine.

In some embodiments, the antiplatelet agent is ibuprofen.

In some embodiments, the antiplatelet agent is vorapaxar.

In some embodiments, the antiplatelet agent is atopaxar.

In some embodiments, the antiplatelet agent is cilostazol.

In some embodiments, the antiplatelet agent is prostaglandin E1.

In some embodiments, the antiplatelet agent is epoprostenol.

In some embodiments, the antiplatelet agent is dipyridamole.

In some embodiments, the antiplatelet agent is treprostinil sodium.

In some embodiments, the antiplatelet agent is sarpogrelate.

In some embodiments, the antiplatelet agent is a supplement.

In some embodiments, the antiplatelet agent is an herbal supplement.

Clotting parameters of blood (e.g., the subject's blood) can be assessed at any appropriate time during the methods described herein. For example, one or more clotting parameters of blood can be assessed before administration of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) such as lyophilized platelets (e.g., anti-fibrinolytic loaded lyophilized platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) as described herein, e.g., in order to determine the need for administration of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) as described herein. As another example, one or more clotting parameters of blood can be assessed after administration of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) as described herein, e.g., in order to determine the effectiveness of the administered composition, to determine whether additional administration of the composition is warranted, or to determine whether it is safe to perform a surgical procedure.

Accordingly, any of the methods described herein can include steps of assessing one or more clotting parameters of blood before administration of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) as described herein, assessing one or more clotting parameters of blood after administration of a composition comprising platelets (e.g., anti-fibrinolytic loaded platelets) or platelet derivatives (e.g., anti-fibrinolytic loaded platelet derivatives) as described herein, or both.

Any appropriate method can be used to assess clotting parameters of blood. Non-limiting examples of methods include the prothrombin time assay, international normalized ratio (INR), thrombin generation (TGA; which can be used to generate parameters such as, e.g., peak thrombin, endogenous thrombin potential (ETP), and lag time), thromboeleastography (TEG), activated clotting time (ACT), and partial thromboplastin time (PTT or aPTT).

INR is a standard method of determining dosing, see equation below, where “PT(x)” is the result of the prothrombin time assay, while the ISI constant is dependent on the manufacturer of the Tissue Factor used in the prothrombin time assay.

INR=((PT(patient))/(PT(normal))){circumflex over ( )}(ISI constant)

Warfarin inhibits the synthesis of four major plasma proteins that are integral to healthy clot formation. A therapeutic maintenance dose of warfarin is typically targeted to an INR of about 2.0 to about 3.0. Thrombosomes present a unique treatment to restore hemostasis in the presence of warfarin-type drugs. Warfarin dose can be expressed by INR, a ratio that increases with the amount of warfarin (1 is a normal value).

In some embodiments, a subject has an INR of more than 2.0 (e.g., at least 2.2, at least 2.4, at least 2.5, at least 2.6, at least 2.8, at least 3.0, at least 3.2, at least 3.4, at least 3.5, at least 3.6, at least 3.8, at least 4.0, at least 4.2, at least 4.4, at least 4.5, at least 4.6, at least 4.8, or at least 5.0) before administration of a composition comprising platelets such as lyophilized platelets or platelet derivatives as described herein. In some embodiments, a subject (e.g., a subject being treated with an anticoagulant, such as warfarin) has an INR of from 2.0 to 3.0, such as from 2.2 to 2.8, such as from 2.4 to 2.6, such as 2.5.

In some embodiments, a subject has a lower INR (or a normal INR) after administration of a composition comprising platelets such as lyophilized platelets or platelet derivatives as described herein. For example, a subject can have an INR of 3.0 or less (e.g., less than 2.8, less than 2.6, less than 2.5, less than 2.4, less than 2.2, less than 2.0, less than 1.8, less than 1.6, less than 1.5, less than 1.4, less than 1.2, or less than 1.0) after administration of a composition comprising platelets or platelet derivatives ad described herein.

Additionally or alternatively, the anti-fibrinolytic loaded platelets can be employed in functional assays. In some embodiments, the anti-fibrinolytic loaded platelets are cold stored, cryopreserved, or lyophilized (to produce thrombosomes) prior to use in therapy or in functional assays.

Any known technique for drying platelets can be used in accordance with the present disclosure, as long as the technique can achieve a final residual moisture content of less than 5%. Preferably, the technique achieves a final residual moisture content of less than 2%, such as 1%, 0.5%, or 0.1%. Non-limiting examples of suitable techniques are freeze-drying (lyophilization) and spray-drying. A suitable lyophilization method is presented in Table A. Additional exemplary lyophilization methods can be found in U.S. Pat. Nos. 7,811,558, 8,486,617, and 8,097,403, each of which are incorporated herein by reference in their entireties. An exemplary spray-drying method includes: combining nitrogen, as a drying gas, with a loading buffer according to the present disclosure, then introducing the mixture into GEA Mobile Minor spray dryer from GEA Processing Engineering, Inc. (Columbia Md., USA), which has a Two-Fluid Nozzle configuration, spray drying the mixture at an inlet temperature in the range of 150° C. to 190° C., an outlet temperature in the range of 65° C. to 100° C., an atomic rate in the range of 0.5 to 2.0 bars, an atomic rate in the range of 5 to 13 kg/hr, a nitrogen use in the range of 60 to 100 kg/hr, and a run time of 10 to 35 minutes. The final step in spray drying is preferentially collecting the dried mixture. The dried composition in some embodiments is stable for at least six months at temperatures that range from −20° C. or lower to 90° C. or higher.

TABLE A Exemplary Lyophilization Protocol Step Temp. Set Type Duration Pressure Set Freezing Step F1 −50° C. Ramp Var N/A F2 −50° C. Hold 3 Hrs N/A Vacuum Pulldown F3 −50° Hold Var N/A Primary Dry P1 −40° Hold 1.5 Hrs 0 mT P2 −35° Ramp 2 Hrs 0 mT P3 −25° Ramp 2 Hrs 0 mT P4 −17° C. Ramp 2 Hrs 0 mT P5 0° C. Ramp 1.5 Hrs 0 mT P6 27° C. Ramp 1.5 Hrs 0 mT P7 27° C. Hold 16 Hrs 0 mT Secondary Dry S1 27° C. Hold >8 Hrs 0 mT

In some embodiments, the step of drying the anti-fibrinolytic loaded platelets that are obtained as disclosed herein, such as the step of freeze-drying the anti-fibrinolytic loaded platelets that are obtained as disclosed herein, includes incubating the platelets with a lyophilizing agent. In some embodiments, the lyophilizing agent is polysucrose. In some embodiments, the lyophilizing agent is a non-reducing disaccharide. Accordingly, in some embodiments, the methods for preparing anti-fibrinolytic loaded platelets further include incubating the anti-fibrinolytic loaded platelets with a lyophilizing agent. In some embodiments, the lyophilizing agent is a saccharide. In some embodiments, the saccharide is a disaccharide, such as a non-reducing disaccharide.

In some embodiments, the step of drying the platelets that are obtained as disclosed herein, such as the step of freeze-drying the platelets that are obtained as disclosed herein, includes incubating the platelets with a lyophilizing agent to generate thrombosomes. In some embodiments, the lyophilizing agent is polysucrose. In some embodiments, the lyophilizing agent is a non-reducing disaccharide. Accordingly, in some embodiments, the methods for preparing thrombosomes from platelets further include incubating the platelets with a lyophilizing agent. In some embodiments, the lyophilizing agent is a saccharide. In some embodiments, the saccharide is a disaccharide, such as a non-reducing disaccharide.

In some embodiments, the platelets are incubated with a lyophilizing agent for a sufficient amount of time and at a suitable temperature to load the platelets with the lyophilizing agent. Non-limiting examples of suitable lyophilizing agents are saccharides, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, and xylose. In some embodiments, non-limiting examples of lyophilizing agent include serum albumin, dextran, polyvinyl pyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In some embodiments, exemplary lyophilizing agents can include a high molecular weight polymer, into the loading composition. By “high molecular weight” it is meant a polymer having an average molecular weight of about or above 70 kDa. Non-limiting examples are polymers of sucrose and epichlorohydrin. In some embodiments, the lyophilizing agent is polysucrose. Although any amount of high molecular weight polymer can be used as a lyophilizing agent, it is preferred that an amount be used that achieves a final concentration of about 3% to 10% (w/v), such as 3% to 7%, for example 6%.

In some embodiments, the process for preparing a composition includes adding an organic solvent, such as ethanol, to the loading solution. In such a loading solution, the solvent can range from 0.1% to 5.0% (v/v).

Within the process provided herein for making the compositions provided herein, addition of the lyophilizing agent can be the last step prior to drying. However, in some embodiments, the lyophilizing agent is added at the same time or before the anti-fibrinolytic, the cryoprotectant, or other components of the loading composition. In some embodiments, the lyophilizing agent is added to the loading solution, thoroughly mixed to form a drying solution, dispensed into a drying vessel (e.g., a glass or plastic serum vial, a lyophilization bag), and subjected to conditions that allow for drying of the solution to form a dried composition.

An exemplary saccharide for use in the compositions disclosed herein is trehalose. Regardless of the identity of the saccharide, it can be present in the composition in any suitable amount. For example, it can be present in an amount of 1 mM to 1 M. In embodiments, it is present in an amount of from 10 mM 10 to 500 mM. In some embodiments, it is present in an amount of from 20 mM to 200 mM. In some embodiments, it is present in an amount from 40 mM to 100 mM. In various embodiments, the saccharide is present in different specific concentrations within the ranges recited above, and one of skill in the art can immediately understand the various concentrations without the need to specifically recite each herein. Where more than one saccharide is present in the composition, each saccharide can be present in an amount according to the ranges and particular concentrations recited above.

The step of incubating the platelets to load them with a cryoprotectant or as a lyophilizing agent includes incubating the platelets for a time suitable for loading, as long as the time, taken in conjunction with the temperature, is sufficient for the cryoprotectant or lyophilizing agent to come into contact with the platelets and, preferably, be incorporated, at least to some extent, into the platelets. In embodiments, incubation is carried out for about 1 minute to about 180 minutes or longer.

The step of incubating the platelets to load them with a cryoprotectant or lyophilizing agent includes incubating the platelets and the cryoprotectant at a temperature that, when selected in conjunction with the amount of time allotted for loading, is suitable for loading. In general, the composition is incubated at a temperature above freezing for at least a sufficient time for the cryoprotectant or lyophilizing agent to come into contact with the platelets. In embodiments, incubation is conducted at 37° C. In certain embodiments, incubation is performed at 20° C. to 42° C. For example, in embodiments, incubation is performed at 35° C. to 40° C. (e.g., 37° C.) for 110 to 130 (e.g., 120) minutes.

In various embodiments, the bag is a gas-permeable bag configured to allow gases to pass through at least a portion or all portions of the bag during the processing. The gas-permeable bag can allow for the exchange of gas within the interior of the bag with atmospheric gas present in the surrounding environment. The gas-permeable bag can be permeable to gases, such as oxygen, nitrogen, water, air, hydrogen, and carbon dioxide, allowing gas exchange to occur in the compositions provided herein. In some embodiments, the gas-permeable bag allows for the removal of some of the carbon dioxide present within an interior of the bag by allowing the carbon dioxide to permeate through its wall. In some embodiments, the release of carbon dioxide from the bag can be advantageous to maintaining a desired pH level of the composition contained within the bag.

In some embodiments, the container of the process herein is a gas-permeable container that is closed or sealed. In some embodiments, the container is a container that is closed or sealed and a portion of which is gas-permeable. In some embodiments, the surface area of a gas-permeable portion of a closed or sealed container (e.g., bag) relative to the volume of the product being contained in the container (hereinafter referred to as the “SA/V ratio”) can be adjusted to improve pH maintenance of the compositions provided herein. For example, in some embodiments, the SA/V ratio of the container can be at least about 2.0 cm²/mL (e.g., at least about 2.1 cm²/mL, at least about 2.2 cm²/mL, at least about 2.3 cm²/mL, at least about 2.4 cm²/mL, at least about 2.5 cm²/mL, at least about 2.6 cm²/mL, at least about 2.7 cm²/mL, at least about 2.8 cm²/mL, at least about 2.9 cm²/mL, at least about 3.0 cm²/mL, at least about 3.1 cm²/mL, at least about 3.2 cm²/mL, at least about 3.3 cm²/mL, at least about 3.4 cm²/mL, at least about 3.5 cm²/mL, at least about 3.6 cm²/mL, at least about 3.7 cm²/mL, at least about 3.8 cm²/mL, at least about 3.9 cm²/mL, at least about 4.0 cm²/mL, at least about 4.1 cm²/mL, at least about 4.2 cm²/mL, at least about 4.3 cm²/mL, at least about 4.4 cm²/mL, at least about 4.5 cm²/mL, at least about 4.6 cm²/mL, at least about 4.7 cm²/mL, at least about 4.8 cm²/mL, at least about 4.9 cm²/mL, or at least about 5.0 cm²/mL. In some embodiments, the SA/V ratio of the container can be at most about 10.0 cm²/mL (e.g., at most about 9.9 cm²/mL, at most about 9.8 cm²/mL, at most about 9.7 cm²/mL, at most about 9.6 cm²/mL, at most about 9.5 cm²/mL, at most about 9.4 cm²/mL, at most about 9.3 cm²/mL, at most about 9.2 cm²/mL, at most about 9.1 cm²/mL, at most about 9.0 cm²/mL, at most about 8.9 cm²/mL, at most about 8.8 cm²/mL, at most about 8.7 cm²/mL, at most about 8.6, cm²/mL at most about 8.5 cm²/mL, at most about 8.4 cm²/mL, at most about 8.3 cm²/mL, at most about 8.2 cm²/mL, at most about 8.1 cm²/mL, at most about 8.0 cm²/mL, at most about 7.9 cm²/mL, at most about 7.8 cm²/mL, at most about 7.7 cm²/mL, at most about 7.6 cm²/mL, at most about 7.5 cm²/mL, at most about 7.4 cm²/mL, at most about 7.3 cm²/mL, at most about 7.2 cm²/mL, at most about 7.1 cm²/mL, at most about 6.9 cm²/mL, at most about 6.8 cm²/mL, at most about 6.7 cm²/mL, at most about 6.6 cm²/mL, at most about 6.5 cm²/mL, at most about 6.4 cm²/mL, at most about 6.3 cm²/mL, at most about 6.2 cm²/mL, at most about 6.1 cm²/mL, at most about 6.0 cm²/mL, at most about 5.9 cm²/mL, at most about 5.8 cm²/mL, at most about 5.7 cm²/mL, at most about 5.6 cm²/mL, at most about 5.5 cm²/mL, at most about 5.4 cm²/mL, at most about 5.3 cm²/mL, at most about 5.2 cm²/mL, at most about 5.1 cm²/mL, at most about 5.0 cm²/mL, at most about 4.9 cm²/mL, at most about 4.8 cm²/mL, at most about 4.7 cm²/mL, at most about 4.6 cm²/mL, at most about 4.5 cm²/mL, at most about 4.4 cm²/mL, at most about 4.3 cm²/mL, at most about 4.2 cm²/mL, at most about 4.1 cm²/mL, or at most about 4.0 cm²/mL. In some embodiments, the SA/V ratio of the container can range from about 2.0 to about 10.0 cm²/mL (e.g., from about 2.1 cm²/mL to about 9.9 cm²/mL, from about 2.2 cm²/mL to about 9.8 cm²/mL, from about 2.3 cm²/mL to about 9.7 cm²/mL, from about 2.4 cm²/mL to about 9.6 cm²/mL, from about 2.5 cm²/mL to about 9.5 cm²/mL, from about 2.6 cm²/mL to about 9.4 cm²/mL, from about 2.7 cm²/mL to about 9.3 cm²/mL, from about 2.8 cm²/mL to about 9.2 cm²/mL, from about 2.9 cm²/mL to about 9.1 cm²/mL, from about 3.0 cm²/mL to about 9.0 cm²/mL, from about 3.1 cm²/mL to about 8.9 cm²/mL, from about 3.2 cm²/mL to about 8.8 cm²/mL, from about 3.3 cm²/mL to about 8.7 cm²/mL, from about 3.4 cm²/mL to about 8.6 cm²/mL, from about 3.5 cm²/mL to about 8.5 cm²/mL, from about 3.6 cm²/mL to about 8.4 cm²/mL, from about 3.7 cm²/mL to about 8.3 cm²/mL, from about 3.8 cm²/mL to about 8.2 cm²/mL, from about 3.9 cm²/mL to about 8.1 cm²/mL, from about 4.0 cm²/mL to about 8.0 cm²/mL, from about 4.1 cm²/mL to about 7.9 cm²/mL, from about 4.2 cm²/mL to about 7.8 cm²/mL, from about 4.3 cm²/mL to about 7.7 cm²/mL, from about 4.4 cm²/mL to about 7.6 cm²/mL, from about 4.5 cm²/mL to about 7.5 cm²/mL, from about 4.6 cm²/mL to about 7.4 cm²/mL, from about 4.7 cm²/mL to about 7.3 cm²/mL, from about 4.8 cm²/mL to about 7.2 cm²/mL, from about 4.9 cm²/mL to about 7.1 cm²/mL, from about 5.0 cm²/mL to about 6.9 cm²/mL, from about 5.1 cm²/mL to about 6.8 cm²/mL, from about 5.2 cm²/mL to about 6.7 cm²/mL, from about 5.3 cm²/mL to about 6.6 cm²/mL, from about 5.4 cm²/mL to about 6.5 cm²/mL, from about 5.5 cm²/mL to about 6.4 cm²/mL, from about 5.6 cm²/mL to about 6.3 cm²/mL, from about 5.7 cm²/mL to about 6.2 cm²/mL, or from about 5.8 cm²/mL to about 6.1 cm²/mL.

Gas-permeable closed containers (e.g., bags) or portions thereof can be made of one or more various gas-permeable materials. In some embodiments, the gas-permeable bag can be made of one or more polymers including fluoropolymers (such as polytetrafluoroethylene (PTFE) and perfluoroalkoxy (PFA) polymers), polyolefins (such as low-density polyethylene (LDPE), high-density polyethylene (HDPE)), fluorinated ethylene propylene (FEP), polystyrene, polyvinylchloride (PVC), silicone, and any combinations thereof.

In some embodiments, the lyophilizing agent as disclosed herein may be a high molecular weight polymer. By “high molecular weight” it is meant a polymer having an average molecular weight of about or above 70 kDa and up to 1,000,000 kDa Non-limiting examples are polymers of sucrose and epichlorohydrin (polysucrose). Although any amount of high molecular weight polymer can be used, it is preferred that an amount be used that achieves a final concentration of about 3% to 10% (w/v), such as 3% to 7%, for example 6%. Other non-limiting examples of lyoprotectants are serum albumin, dextran, polyvinyl pyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In some embodiments, a lyoprotectant is also a cryoprotectant. For example, albumin, polysucrose, and sucrose can also be used as a cryoprotectant.

In some embodiments, lyophilized platelets can be fixed (e.g., lyophilized fixed plates) in fixing agent. In some embodiments, lyophilized platelets can be fixed in formalin (e.g., lyophilized formalin-fixed platelets).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1,000 k/μl to about 500,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 5,000 k/μl to about 450,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 10,000 k/μl to about 400,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 30,000 k/μl to about 300,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 40,000 k/μl to about 250,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 50,000 k/μl to about 225,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 60,000 k/μl to about 200,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 70,000 k/μl to about 175,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 80,000 k/μl to about 150,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 90,000 k/μl to about 125,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 100,000 k/μl to about 120,000 k/μl. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 105,000 k/μl to about 115,000 k/μl. In some embodiments, the therapeutically effective amount of lyophilized platelets (e.g., thrombosomes) can be at any of the concentrations described herein).

In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1×10² particles/kg to from about 1×10¹³ particles/kg. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1×10³ particles/kg to from about 1×10¹² particles/kg. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1×10⁴ particles/kg to from about 1×10¹¹ particles/kg. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1×10⁵ particles/kg to from about 1×10¹⁰ particles/kg. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1×10⁶ particles/kg to from about 1×10⁹ particles/kg. In some embodiments, the lyophilized platelets (e.g., thrombosomes) can be at a concentration from about 1×10⁷ particles/kg to from about 1×10⁸ particles/kg. In some embodiments, a therapeutically effective amount of the lyophilized platelets (e.g., thrombosomes) can be at any of the concentrations described herein.

In some embodiments of the methods herein, any of the compositions described herein are administered topically. In some embodiments, topical administration can include administration via a solution, cream, gel, suspension, putty, particulates, or powder. In some embodiments, topical administration can include administration via a bandage (e.g. an adhesive bandage or a compression bandage) or medical closure (e.g., sutures, staples)); for example the anti-fibrinolytic loaded platelet derivatives (e.g., lyopreserved platelets (e.g., thrombosomes)) can be embedded therein or coated thereupon), as described in PCT Publication No. WO2017/040238 (e.g., paragraphs [013]-[069]), corresponding to U.S. patent application Ser. No. 15/776,255, the entirety of which is herein incorporated by reference.

In some embodiments of the methods herein, the compositions described herein are administered parenterally.

In some embodiments of the methods herein, the compositions described herein are administered intravenously.

In some embodiments of the methods herein, the compositions described herein are administered intramuscularly.

In some embodiments of the methods herein, the compositions described herein are administered intrathecally.

In some embodiments of the methods herein, the compositions described herein are administered subcutaneously.

In some embodiments of the methods herein, the compositions described herein are administered intraperitoneally. In some embodiments, the anti-fibrinolytic loaded platelets prepared as disclosed herein have a storage stability that is at least about equal to that of the platelets prior to the loading of the anti-fibrinolytic.

The loading buffer may be any buffer that is non-toxic to the platelets and provides adequate buffering capacity to the solution at the temperatures at which the solution will be exposed during the process provided herein. Thus, the buffer may include any of the known biologically compatible buffers available commercially, such as phosphate buffers, such as phosphate buffered saline (PBS), bicarbonate/carbonic acid, such as sodium-bicarbonate buffer, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and tris-based buffers, such as tris-buffered saline (TBS). Likewise, it may include one or more of the following buffers: propane-1,2,3-tricarboxylic (tricarballylic); benzenepentacarboxylic; maleic; 2,2-dimethylsuccinic; 3,3-dimethylglutaric; bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS); benzenehexacarboxylic (mellitic); N-(2-acetamido)imino-diacetic acid (ADA); butane-1,2,3,4-tetracarboxylic; pyrophosphoric; 1,1-cyclopentanediacetic (3,3 tetramethylene-glutaric acid); piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES); N-(2-acetamido)-2-amnoethanesulfonic acid (ACES); 1,1-cyclohexanediacetic; 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid (EMTA; ENDCA); imidazole; 2-(aminoethyl)trimethylammonium chloride (CHOLAMINE); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 2-methylpropane-1,2,3-triscarboxylic (beta-methyltricarballylic); 2-(N-morpholino)propane-sulfonic acid (MOPS); phosphoric; and N-tris(hydroxymethyl)methyl-2-amminoethane sulfonic acid (TES).

A plate reader (e.g., Tecan Microplate reader (e.g., Infinite® 200 PRO)) can be used to quantify loading efficiency of the anti-fibrinolytic in the anti-fibrinolytic loaded platelets. Platelets can be evaluated for functionality by adenosine diphosphate (ADP), collagen, arachidonic acid, phorbol myristate acetate (PMA), thrombin receptor activating peptide (TRAP), and/or any other platelet agonist known in the art for stimulation post-loading. A hemostasis analyzer (e.g., TEG® 5000 Thromboelastogram® Hemostasis Analyzer system) can be used to test anti-fibrinolytic function of EACA loaded platelets.

In some embodiments, the anti-fibrinolytic platelets are lyophilized. In some embodiments, the anti-fibrinolytic loaded platelets are cryopreserved. In some embodiments, the unloaded platelets are lyophilized. In some embodiments, the unloaded platelets are cryopreserved.

In some embodiments, the anti-fibrinolytic loaded platelets retain the loaded anti-fibrinolytic compound upon rehydration and release the anti-fibrinolytic compound upon stimulation by endogenous platelet activators, such as endogenous platelet activators described herein.

In some embodiments, the dried platelets (such as freeze-dried platelets) retain the loaded anti-fibrinolytic upon rehydration and release the anti-fibrinolytic (e.g., EACA) upon stimulation by endogenous platelet activators. In some embodiments, at least about 10%, such as at least about 20%, such as at least about 30% of the anti-fibrinolytic is retained. In some embodiments, from about 10% to about 20%, such as from about 20% to about 30% of the anti-fibrinolytic is retained.

In some embodiments, anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes can shield the anti-fibrinolytic from exposure in circulation, thereby reducing or eliminating systemic toxicity (e.g. cardiotoxicity) associated with the anti-fibrinolytic. In some embodiments, anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, and/or anti-fibrinolytic loaded thrombosomes can also protect the anti-fibrinolytic from metabolic degradation or inactivation. In some embodiments, anti-fibrinolytic delivery with anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, and/or anti-fibrinolytic loaded thrombosomes can therefore be advantageous in treatment of diseases such as traumatic bleeding events (e.g., hemorrhage) and/or hemophilia, since anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, and/or anti-fibrinolytic loaded thrombosomes can mitigate systemic side effects. In some embodiments, anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, and/or anti-fibrinolytic loaded thrombosomes can be used in any therapeutic setting in which expedited healing process is required or advantageous.

In some embodiments, provided herein is a method of treating a disease as disclosed herein in a subject in need thereof, comprising administering anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes as disclosed herein. In some embodiments, provided herein is a method of treating a disease as disclosed herein in a subject in need thereof, comprising administering cold stored, room temperature stored, cryopreserved, thawed, rehydrated, and/or lyophilized platelets, platelet derivatives, or thrombosomes as disclosed herein. In some embodiments, the disease is inherited (e.g., congenital, classic) hemophilia. In some embodiments, the inherited hemophilia is hemophilia A. In some embodiments, the inherited hemophilia is hemophilia B.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising providing platelets and contacting the platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are contacted with the anti-fibrinolytic and with the loading buffer sequentially, in either order.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising contacting platelets with the anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising contacting the platelets with a buffer including a salt, a base, a loading agent, and optionally at least one organic solvent to form a first composition and contacting the first composition with an anti-fibrinolytic, to form the anti-fibrinolytic loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are contacted with the anti-fibrinolytic and with the loading buffer concurrently.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising contacting the platelets with an anti-fibrinolytic in the presence of a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent to form the anti-fibrinolytic-loaded platelets. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are pooled from a plurality of donors prior to a treating step.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition and contacting the first composition with an anti-fibrinolytic to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets with an anti-fibrinolytic in the presence of a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising contacting thrombosomes with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes prepared by a process comprising providing platelets and contacting the platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the anti-fibrinolytic loaded thrombosomes. In some embodiments of preparing anti-fibrinolytic loaded platelets, the platelets are contacted with the anti-fibrinolytic and with the loading buffer sequentially, in either order.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising contacting platelets with the anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising contacting platelets with a buffer including a salt, a base, a loading agent, and optionally at least one organic solvent to form a first composition and contacting the first composition with an anti-fibrinolytic, and a freeze drying step, to form the anti-fibrinolytic loaded thrombosomes. In some embodiments of preparing anti-fibrinolytic loaded thrombosomes, the platelets are contacted with the anti-fibrinolytic and with the loading buffer concurrently.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising contacting platelets with an anti-fibrinolytic in the presence of a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying to form the anti-fibrinolytic-loaded thrombosomes. In some embodiments of preparing anti-fibrinolytic loaded thrombosomes, the platelets are pooled from a plurality of donors prior to a treating step.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a freeze-drying step, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an anti-fibrinolytic to form a first composition and contacting the first composition with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition and contacting the first composition with an anti-fibrinolytic, and a step of freeze-drying to form the anti-fibrinolytic loaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic loaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets with an anti-fibrinolytic in the presence of a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the anti-fibrinolytic loaded thrombosomes.

In some embodiments, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be advantageous in the treatment of diseases such as a hemophilia (e.g., congenital hemophilia). In some embodiments, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be advantageous in the treatment of disease such as congenital hemophilia.

In some embodiments, provided herein is a method of treating a disease as disclosed herein in a subject in need thereof, (e.g., congenital hemophilia A, congenital hemophilia B), comprising administering to a subject in need thereof, unloaded platelets, unloaded platelet derivatives, or unloaded thrombosomes as disclosed herein. In some embodiments, provided herein is a method of treating a disease as disclosed herein in a subject in need thereof, comprising administering unloaded cold stored, room temperature stored, cryopreserved thawed, rehydrated, and/or lyophilized platelets, unloaded platelet derivatives, or unloaded thrombosomes as disclosed herein.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising contacting thrombosomes with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes prepared by a process comprising providing platelets and contacting the platelets with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising contacting platelets with a loading buffer including a salt and a base to form a first composition and contacting the first composition with a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising contacting platelets with a loading agent, and optionally at least one organic solvent to form a first composition and contacting the first composition with a loading buffer including a salt and a base, and a freeze-drying step, to form the unloaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising contacting platelets a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a step of freeze-drying to form the anti-fibrinolytic-loaded thrombosomes. In some embodiments of preparing unloaded thrombosomes, the platelets are pooled from a plurality of donors prior to a treating step.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, and a freeze-drying step, to form the unloaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with a loading buffer including a salt and a base to form a first composition and contacting the first composition with a loading agent, and optionally at least one organic solvent, and a step of freeze-drying, to form the unloaded thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of unloaded thrombosomes, wherein the unloaded thrombosomes are prepared by a process comprising A) pooling platelets from a plurality of donors and B) contacting the platelets from step (A) with an a loading agent to form a first composition and contacting the first composition with a loading buffer including a salt and a base, and optionally at least one organic solvent, and a step of freeze-drying, to form the unloaded thrombosomes.

In some embodiments, no solvent is used. Thus, provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;     -   B) contacting the platelets with an anti-fibrinolytic and with a         loading buffer comprising a salt, a base, and a loading agent,         to form the anti-fibrinolytic loaded platelets,     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol, and     -   C) a step of freeze-drying, to form the anti-fibrinolytic loaded         thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;     -   B) contacting the platelets with an anti-fibrinolytic to form a         first composition;     -   C) contacting the first composition with a buffer comprising a         salt, a base, and a loading agent, to form the anti-fibrinolytic         loaded platelets,     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol and the method does not         comprise contacting the first composition with an organic         solvent such as ethanol, and     -   (D) a step freeze-drying, to form the anti-fibrinolytic loaded         thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) isolating platelets, for example in a liquid medium;     -   B) contacting the platelets with a buffer comprising a salt, a         base, and a loading agent, to form a first composition;     -   C) contacting the first composition with an anti-fibrinolytic,         to form the anti-fibrinolytic loaded platelets,     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol and the method does not         comprise contacting the first composition with an organic         solvent such as ethanol and     -   D) a step of freeze drying, to form the anti-fibrinolytic loaded         thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   A) preparing platelets;     -   B) contacting the platelets with an anti-fibrinolytic and with a         loading buffer comprising         -   a salt, a base, and a loading agent, to form the             anti-fibrinolytic loaded platelets,         -   wherein the method does not comprise contacting the             platelets with an organic solvent such as ethanol, and     -   C) a step of freeze-drying, to form the anti-fibrinolytic loaded         thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   a) preparing platelets;     -   b) contacting the platelets with an anti-fibrinolytic to form a         first composition;     -   c) contacting the first composition with a buffer comprising a         salt, a base, and a loading agent, to form the anti-fibrinolytic         loaded platelets,     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol and the method does not         comprise contacting the first composition with an organic         solvent such as ethanol and     -   d) a step of freeze-drying, to form the anti-fibrinolytic loaded         thrombosomes.

Provided herein are methods to treat congenital hemophilia (e.g., congenital hemophilia A or congenital hemophilia B), comprising a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes, wherein the anti-fibrinolytic thrombosomes are prepared by a process comprising:

-   -   a) preparing platelets;     -   b) contacting the platelets with a buffer comprising a salt, a         base, and a loading agent, to form a first composition;     -   c) contacting the first composition with an anti-fibrinolytic,         to form the anti-fibrinolytic loaded platelets.     -   wherein the method does not comprise contacting the platelets         with an organic solvent such as ethanol and the method does not         comprise contacting the first composition with an organic         solvent such as ethanol and     -   d) a freeze-drying step, to form the anti-fibrinolytic loaded         thrombosomes.

In some embodiments, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be advantageous in the treatment of diseases such as a hemophilia. In some embodiments, unloaded platelets, unloaded platelet derivatives, and/or unloaded thrombosomes can be advantageous in the treatment of disease such as classic hemophilia (e.g., inherited).

In some embodiments, provided herein is a method of treating a disease as disclosed herein in a subject in need thereof, (e.g., congenital hemophilia, comprising administering to a subject in need thereof, unloaded platelets, unloaded platelet derivatives, or unloaded thrombosomes as disclosed herein. In some embodiments, provided herein is a method of treating a disease as disclosed herein in a subject in need thereof, comprising administering unloaded cold stored, room temperature stored, cryopreserved thawed, rehydrated, and/or lyophilized platelets, unloaded platelet derivatives, or unloaded thrombosomes as disclosed herein.

In some embodiments, provided herein are methods of treatment of a disease with anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes as disclosed herein. For example, the treatment of a disease can be performed in a model for a disease (e.g., Hemophilia A, Hemophilia B). For example, plasma chemically depleted of Factor VIII (e.g., plasma substantially similar to plasma from a subject with hemophilia A) can be treated with anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes as disclosed herein. For example, plasma immuno-depleted of Factor IX (e.g., plasma substantially similar to plasma from a subject with hemophilia B) can be treated with anti-fibrinolytic loaded platelets, anti-fibrinolytic loaded platelet derivatives, or anti-fibrinolytic loaded thrombosomes as disclosed herein.

In some embodiments, provided herein are methods of treatment of a disease including administering to a subject in need thereof, unloaded platelets, unloaded platelet derivatives, or unloaded thrombosomes as disclosed herein. For example, the treatment of a disease can be performed in a model for a disease (e.g., Hemophilia A, Hemophilia B). For example, plasma chemically depleted of Factor VIII (e.g., plasma substantially similar to plasma from a subject with hemophilia A) can be treated with unloaded platelets, unloaded platelet derivatives, or unloaded thrombosomes. For example, plasma immuno-depleted of Factor IX (e.g., plasma substantially similar to plasma from a subject with hemophilia B) can be treated with unloaded platelets, unloaded platelet derivatives, or unloaded thrombosomes.

In some embodiments, provided herein are methods of treatment of a disease as disclosed herein, including administering unloaded cold stored, room temperature stored, cryopreserved thawed, rehydrated, and/or lyophilized platelets, unloaded platelet derivatives, or unloaded thrombosomes as disclosed herein to a model of a disease (e.g., Hemophilia A, Hemophilia B).

A loading agent (e.g., an incubating agent) can include any appropriate components. In some embodiments, the loading agent may comprise a liquid medium. In some embodiments the loading agent may comprise one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salt that can be found in blood or blood products, or that is known to be useful in drying platelets (e.g., anti-fibrinolytic loaded platelets), or any combination of two or more of these.

In some embodiments, provided herein is composition comprising platelets such as lyophilized platelets or platelet derivatives (e.g., thrombosomes), polysucrose and trehalose made by the process comprising obtaining fresh platelets, optionally incubating the platelets in DMSO, isolating the platelets by centrifugation, resuspending the platelets in an incubating agent which comprises trehalose and ethanol thereby forming a first mixture, incubating the first mixture, mixing polysucrose with the first mixture, thereby forming a second mixture, and lyophilizing the second mixture to form a freeze dried composition comprising platelets or platelet derivatives (e.g., thrombosomes), polysucrose and trehalose.

In some embodiments, provided herein is a method of making a freeze-dried platelet composition comprising platelets or platelet derivatives (e.g., thrombosomes), polysucrose and trehalose comprising obtaining fresh platelets, optionally incubating the platelets in DMSO, isolating the platelets by centrifugation, resuspending the platelets in a incubating agent which comprises trehalose and ethanol thereby forming a first mixture, incubating the first mixture, mixing polysucrose with the first mixture, thereby forming a second mixture, and lyophilizing the second mixture to form a freeze-dried composition comprising platelets or platelet derivatives (e.g., thrombosomes), polysucrose and trehalose.

In some embodiments, provided herein is a process for making freeze-dried platelets, the process comprising incubating isolated platelets in the presence of at least one saccharide under the following conditions: a temperature of from 20° C. to 42° C. for about 10 minutes to about 180 minutes, adding to the platelets at least one cryoprotectant, and lyophilizing the platelets, wherein the process optionally does not include isolating the platelets between the incubating and adding steps, and optionally wherein the process does not include exposing the platelets to a platelet activation inhibitor. The cryoprotectant can be a polysugar (e.g., polysucrose). The process can further include heating the lyophilized platelets at a temperature of 70° C. to 80° C. for 8 to 24 hours. The step of adding to the platelets at least one cryoprotectant can further include exposing the platelets to ethanol. The step of incubating isolated platelets in the presence of at least one saccharide can include incubating in the presence of at least one saccharide. The step of incubating isolated platelets in the presence of at least one saccharide can include incubating in the presence of at least one saccharide. The conditions for incubating can include incubating for about 100 minutes to about 150 minutes. The conditions for incubating can include incubating for about 110 minutes to about 130 minutes. The conditions for incubating can include incubating for about 120 minutes. The conditions for incubating can include incubating at 35° C. to 40° C. The conditions for incubating can include incubating at 37° C. The conditions for incubating can include incubating at 35° C. to 40° C. for 110 minutes to 130 minutes. The conditions for incubating can include incubating at 37° C. for 120 minutes. The at least one saccharide can be trehalose, sucrose, or both trehalose and sucrose. The at least one saccharide can be trehalose. The at least one saccharide can be sucrose.

In some embodiments, provided herein is a method of preparing freeze-dried platelets, the method including providing platelets, suspending the platelets in a salt buffer that includes about 100 mM trehalose and about 1% (v/v) ethanol to make a first composition, incubating the first composition at about 37° C. for about 2 hours, adding polysucrose (e.g., polysucrose 400) to a final concentration of about 6% (w/v) to make a second composition, lyophilizing the second composition to make freeze-dried platelets, and heating the freeze-dried platelets at 80° C. for 24 hours.

While the embodiments of the methods and compositions described herein are amenable to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are described in detail below. The intention, however, is not to limit the methods and compositions to the particular embodiments described. On the contrary, the methods and compositions are intended to cover all modifications, equivalents, and alternatives falling within the scope of the methods and compositions as defined by the appended claims.

EXAMPLES Example 1. Loading Platelets with ε-Aminocaproic Acid (EACA)

Protocol 1

The starting apheresis platelet material was pooled and acidified to a pH 6.6-6.8 using 4 μL of 1M Acid Citrate Dextrose solution per 1 ml of pooled platelet rich plasma. A count of platelets was obtained in solution using a Coulter AcT Diff hematology analyzer. The platelets were isolated via centrifugation at 1500 g×for 20 minutes at room temperature with gentle acceleration and braking.

A dansyl-EACA/EACA solution was prepared as follows: stock of 1 mM dansyl-EACA and 1M EACA (1:1000 ratio), dissolved in loading buffer, and frozen. The frozen dansyl-EACA/EACA solution was thawed at 37° C. for 20 minutes.

The platelets were resuspended in loading buffer at a concentration of 2,250,000 platelets/μL. The platelets (2,250,000 platelets/μl) were combined with the EACA solution as follows: 9 ml platelets at 2,250,000 platelets/μL and 1 ml dansyl-EACA/EACA stock as prepared above and incubated at 37° C. for 3 hours. 1 mL of dansyl-EACA/EACA stock and 30 μL of 1 M dextrose were supplemented every hour on the hour.

The EACA-loaded platelets were cryopreserved by incubating the samples in a freezer at −80° C. The cryopreserved EACA-loaded platelets were thawed at 37° C. water bath and used for downstream applications described herein. A Tecan Infinite M200 Pro plate reader was used for quantification of anti-fibrinolytic loading. A TEG 5000 Hemostasis analyzer was used to evaluate anti-fibrinolytic function of EACA from EACA loaded platelets.

TABLE 1 Loading Buffer with EACA Concentration Component (mM, unless specified otherwise) NaCl 750 KCl 48 HEPES 95 NaHCO₃ 120 Dextrose 3 Trehalose 0.1 Dansyl-EACA/EACA 0, 50, or 100 (1:1000)

FIG. 1 is a graph showing dose-dependent EACA loading into platelets at 50 mM and 100 mM over time measured at 1 hour, 2 hours, 3 hours, and 4 hours. The pooled apheresis platelets were incubated with dansyl-EACA (λex/em: 325 nm, 570 nm) and unlabeled EACA at a molar ratio of 1:1000 in loading buffer. Platelets were incubated for 1, 2, 3, or 4 hours at 37° C. with low frequency agitation on a rocker. After isolation by centrifugation and washing, the platelets were lysed by sonication and EACA per platelet was quantified using the Tecan Infinite® M200 PRO plate reader. The results show dose-dependent and time-dependent EACA loading into platelets as measured by the concentration (mg/platelet) of EACA per platelet in each sample.

Example 2. EACA-Loaded Platelet Functionality

FIG. 2 shows in vitro agonist stimulation of EACA release from EACA-loaded platelets with known agonists: phorbol myristate acetate (PMA), collagen, or thrombin receptor activating peptide (TRAP) agonists. Platelets were loaded with EACA according to Protocol 1. EACA loaded platelets were incubated in either 1 μg/mL PMA, 10 μg/mL collagen, or 10 μM TRAP in HMT buffer with 1 mM MgCl₂ for 10 minutes at 37° C. to stimulate EACA release. After incubation, the platelets were isolated as a pellet by centrifugation at 1470 g×10 minutes. The released (supernatant) and intracellular (pellet) EACA concentrations were quantified using the Tecan Infinite® M200 PRO plate reader.

FIG. 3 is a graph showing an EACA dose-response curve in pooled human platelet rich plasma to determine the effect of free EACA on lysis after 30 minutes. Tissue plasminogen activator was used to induce fibrinolysis in vitro. 690 μL platelet rich plasma (platelets in George King plasma) was mixed with 10 μL of 65 μg/mL tPA and 10 μL of serially diluted samples of EACA in cell culture grade water. Each measurement was run in duplicate using 340 μL of the EACA/tPA mixture pipetted into 20 μL of 0.2 M CaCl₂). Runs were maintained for 30 minutes after Maximum Amplitude (MA) had been reached at 37° C. The percent lysis (LY30) shows the extent of fibrinolysis 30 minutes after MA had been reached. A LY30 of 100% corresponds to complete lysis of the blood clot while 0% corresponds to the absence of lysis. 140 μg/mL of free EACA was shown to effectively inhibit fibrinolysis (LY30 of 3.5%).

FIGS. 4A-E shows that EACA-loaded platelets can release EACA in vitro to prevent fibrinolysis. FIGS. 4A-E show thromboelastogram (TEG) graphs of EACA loaded platelets with tissue plasminogen activator at varying platelet concentrations. Platelet concentrations tested were 125 kcell/μL, 250 kcell/μL, 500 kcell/μL, 1000 kcell/μL, and 2000 kcell/μL of EACA-loaded platelets. Maximum amplitude was maintained over 30 minutes with increasing concentration of drug loaded platelets. FIG. 4F shows a dose-response curve of EACA-loaded platelets. The platelets were lysed and the concentration of EACA for each individual dose from A-E is plotted on the x-axis. A dose of EACA-loaded platelets equivalent to 2.5 μg/mL prevents fibrinolysis (LY30 of 5.6%). 140 μg/mL free EACA inhibit fibrinolysis to a similar extent (LY30 of 3.5%). Therefore, EACA loaded into platelets can inhibit tPA-induced coagulopathy at 56× lower concentrations than the free EACA.

FIG. 5 is a graph comparing the percent lysis of clots at 30 minutes for free EACA in solution (FIG. 3) and EACA-loaded platelets (FIGS. 4A-E). FIG. 5 shows, on a log scale, approximately equal percent lysis is observed using platelet-loaded EACA at concentrations between 17 and 56-fold lower than for non-loaded EACA (free EACA). These results show that platelets are a viable vehicle for delivery of an anti-fibrinolytic such as EACA at low concentrations relative to free EACA.

Example 3. Cryopreserved EACA-Loaded Platelets

FIG. 6 is a graph measuring the amount of EACA mg/platelet of EACA-loaded platelets pre-cryopreservation and post-cryopreservation. Platelets were incubated in 100 mM dansyl-EACA (λex/em: 325 nm, 570 nm) and unlabeled EACA at a molar ratio of 1:1000 in loading buffer (Table 1) for 3 hours followed by centrifugation and resuspension in loading buffer containing sucrose and minimal DMSO for cryopreservation. The samples were cryopreserved by incubation of the samples in a −80° C. freezer. Drug load of pre- and post-cryopreservation were quantified using a Tecan Infinite M200 Pro plate reader. The cryopreserved platelets retained 91% of the original amount of EACA that was loaded per cell before preservation.

FIGS. 7A-E shows that cryopreserved EACA-loaded platelets (EACA-loaded platelets in the presence of loading buffer (Table 1) can release EACA in vitro to prevent fibrinolysis. FIGS. 7A-D show TEG graphs of cryopreserved EACA loaded platelets plus tissue plasminogen activator at varying platelet concentrations. Platelet concentrations ranged from 100 kcell/μL, 250 kcell/μL, 500 kcell/μL, and 1000 kcell/μL. MA was maintained after 30 minutes with increasing concentration of cryopreserved drug loaded platelets. FIG. 7E shows a dose-response curve of cryopreserved EACA loaded platelets. The platelets were lysed and the concentration of EACA for each individual dose from A-E is plotted on the x-axis. A dose of EACA-loaded platelets equivalent to 5 μg/mL prevents fibrinolysis (LY30 of 2.1%). 140 μg/mL free EACA inhibit fibrinolysis to a similar extent (LY30 of 3.5%). Therefore, EACA loaded into platelets can inhibit tPA-induced coagulopathy at 28× lower concentrations than free EACA.

FIGS. 8A-C show graphs indicating the strength of clots as measured by maximum amplitude (MA). FIG. 8A shows MA measured in the presence of free EACA. FIGS. 8B-C shows MA measured with EACA loaded platelets (5×10⁵ platelets/μl) in the presence of loading buffer pre-cryopreservation (8B) and post-cryopreservation (8C). MA increases with increasing number of EACA-loaded platelets in both pre-cryopreservation and post-cryopreservation.

Example 4 Thrombosomes Enhance Peak Thrombin Concentration and Total Amount (ETP) Production in Hemophilic B Plasma

Hemophilic B plasma is plasma that lacks about 95% of Factor IX. FIGS. 9-11 show that thrombosomes enhance the peak thrombin concentration and the total amount of (ETP) production in hemophilic B plasma.

Normal platelet poor plasma (George King pooled plasma) was compared to Factor IX deficient plasma in the thrombin generation assay. Assessment of the start of production (lag time), peak production (peak thrombin concentration), and the amount of time to peak thrombin production, as well as, total possible thrombin was collected for each sample type. The samples were stimulated to start the clotting cascade with a PPP reagent (low amounts of tissue factor and phospholipids) as supplied by the manufacturer. The lag time and time to peak for normal plasma and Factor IX deficient plasma were similar, though the peak thrombin and thrombin potential were much lower in the Factor IX deficient plasma. When Factor IX deficient plasma was supplemented with 75 k/μL of thrombosomes the thrombin potential mostly recovered to normal and the peak thrombin partially returns to normal (FIGS. 9-11). FIG. 9 shows a histogram of thrombin production after clot initiation. George King normal pooled plasma demonstrated a normal thrombin profile. Factor IX deficient plasma decreased thrombin production parameters, however, the addition of 75 k/μL of thrombosomes partially recovered the Factor IX deficient plasma to a normal profile. FIG. 10 shows that depression of thrombin production in Factor IX deficient plasma was partially recovered with thrombosomes. Peak thrombin production was reduced in Factor IX deficient plasma and partially recovered with the addition of 75 k/μL thrombosomes. FIG. 11 shows a loss of endogenous thrombin potential (ETP) in Factor IX deficient plasma is partially recovered with the addition of thrombosomes (75 k/μL).

Example 5—EACA-Loaded Fresh Platelets can Partially Restore Hemostasis in Congenital Hemophilia A and B

Table 2 summarizes numeric thromboelastography data from both congenital models of Hemophilia A and Hemophilia B. The first row is a positive control of George King Plasma. Rows 2-4 show numeric thromboelastography data from platelet rich plasma models of congenital Hemophilia B of varying depletion percentages. For example, row 2 shows a platelet rich plasma model of congenital Hemophilia B completely deficient in coagulation Factor IX (100% depleted), row 3 shows a platelet rich plasma model of congenital Hemophilia B 99% deficient in coagulation Factor IX, and row 4 shows a platelet rich plasma model of congenital Hemophilia B 90% deficient in coagulation Factor IX. Row 5 shows numeric thromboelastography data from a platelet rich plasma model of congenital Hemophilia A completely deficient (100%) in coagulation Factor VIII.

TABLE 2 R time (sec) K time (sec) α (slope) Control 585 100 70 100% Factor IX 4775 2035 NA * DEPLETED 99% Factor IX 1775 493 32 DEPLETED 90% Factor IX 1350 307 46 DEPLETED 100% Factor VIII 915 163 61.6 DEPLETED

FIGS. 12A-E show fresh platelets loaded with EACA partially restored hemostasis in a plasma model of congenital Hemophilia A (100% Factor VIII deficient). FIG. 12A shows a model thromboelastography graph in George King Plasma (positive control) and FIG. 12C shows a thromboelastography graph of plasma in a model of congenital Hemophilia A. Both George King Plasma and congenital Hemophilia A model platelet rich plasma were treated with 140 μg/mL of free EACA (FIGS. 12C and 12D, respectively). The data show that free EACA on George King Plasma did not affect hemostasis and free EACA in the congenital Hemophilia A plasma model did not improve numeric thromboelastography metrics (Table 3). In contrast, FIG. 12E shows restoration of hemostasis consistent with the positive control (FIG. 12A) when the platelet rich plasma model of congenital Hemophilia A was treated with fresh platelets loaded with EACA. Thus, the data in FIGS. 12A-E and Table 3 below demonstrate that fresh platelets loaded with EACA are more effective at restoring hemostasis in a platelet rich plasma model of congenital Hemophilia A (100% Factor VIII deficient) than free EACA alone.

Table 3 below summarizes the numeric thromboelastography data from platelet rich plasma models of congenital Hemophilia A and B (PRP*=platelet rich plasma at 400,000 cells/μL).

TABLE 3 R time (sec) K time (sec) α (slope) Control PRP * 585 100 69.5 100% Factor VIII 915 163 61.6 DEPLETED PRP 100% Factor VIII 1030 285 44.8 DEPLETED PRP + FREE EACA (140 μg/ml) 100% Factor VIII 363 128 67 DEPLETED PRP + LOADED EACA (5 μg/ml)

Table 4 summarizes numeric thromboelastography data from a platelet rich plasma model of congenital Hemophilia B (100% Factor IX deficient). Rows 1 and 2 are positive and negative controls, respectively. The data in the third row show the results of platelet rich plasma model of congenital Hemophilia B treated with 1406 g/mL of free EACA and the data in the fourth row show the results of platelet rich plasma of congenital Hemophilia B treated with fresh platelets loaded with EACA (5 μg/mL).

The data in rows three and four of Table 4 show that EACA loaded fresh platelets are more effective than free EACA alone in a plasma rich platelet model of congenital Hemophilia B (100% Factor IX deficient)(PRP*=platelet rich plasma at 400,000 cell/L).

TABLE 4 R time (sec) K time (sec) α (slope) Control PRP * 585 100 69.5 100% Factor IX 4775 2035 unmeasurable DEPLETED PRP 100% Factor IX 2023 478 30.7 DEPLETED PRP + FREE EACA (140 μg/mL) 100% Factor IX 418 223 49.9 DEPLETED PRP+ LOADED EACA (5 μg/mL)

Example 6—Thrombin Generation in Immunodepleted Factor IX Plasma

Immunodepletion of coagulation Factor IX from platelet rich plasma is another way to generate a platelet rich plasma model of Hemophilia B (Factor IX deficient plasma). FIGS. 13, 14, and 15 show thrombin generation measured (nM) under various conditions in 100%, 99%, and 90% immunodepleted Factor IX (Hemophilia B) plasma over time, respectively. The platelet rich plasma in all three experiments included 400,000 cells/μL and a positive control of George King Plasma (GKP). All three experiments also included a control of coagulation Factor IX immunodepleted plasma alone (FIGS. 13, 14, and 15 included a control of immunodepleted Factor IX of 100%, 99%, and 90%, respectively).

Each immunodepleted Factor IX platelet rich plasma sample (100%, 99%, and 90%) were independently treated with either free EACA (400 μg/mL) alone or fresh platelets loaded with EACA (4.7 μg). FIG. 14 (100% Factor IX deficient) and FIG. 16 (90% Factor IX deficient) show that free EACA had no effect on the platelet rich plasma's ability to generate thrombin. The data in FIG. 15 (99% Factor IX deficient) show that free EACA had a negative effect on the platelet rich plasma ability to generate thrombin. In contrast, fresh platelets loaded with EACA (4.7 μg) improved thrombin generation (nM) over free EACA alone in both 100% (FIG. 14) and 99% (FIG. 15) immunodepleted Factor IX platelet rich plasma. The data show in FIG. 16 (90% Factor IX deficient) that fresh platelets loaded with EACA restore thrombin generation (nM) to nearly the same concentration as the positive control (George King Plasma). Thus, fresh platelets loaded with EACA are more effective at improving thrombin generation in immunodepleted Factor IX (100%, 99%, and 90%) platelet rich plasma than free EACA alone. Moreover, the concentration of EACA (4.7 μg) in the loaded fresh platelets was significantly less than the concentration tested with free EACA (400 μg/mL).

The data in FIG. 16 show peak thrombin generation (nM) in a platelet rich plasma model of either 99% immunodepleted Factor IX (Hemophilia B) or 90% immunodepleted Factor IX. The first, second, and fifth columns shows the results from untreated platelet rich plasma (George King Plasma), untreated 99% immunodepleted Factor IX plasma, and untreated 90% immunodepleted Factor IX plasma and function as controls. Each plasma was either treated with free EACA (400 μg/mL) or fresh platelets loaded with EACA (5 μg/mL). The data demonstrate that free EACA had little to no effect on either the 99% (column 3) or the 90% (column 6) immunodepleted Factor IX plasmas' peak thrombin generation. In contrast, fresh platelets loaded with EACA (5 μg/mL) restored peak thrombin generation in both the 99% and the 90% immunodepleted Factor IX platelet rich plasma. A larger increase in peak thrombin generation was measured in the 90% immunodepleted Factor IX platelet rich plasma relative to the peak thrombin generation measured in the 99% immunodepleted platelet rich plasma.

The data in FIG. 17 show restoration of total thrombin generation (nM) in a platelet rich plasma model of either 99% immunodepleted Factor IX (Hemophilia B) or 90% immunodepleted factor IX. The first, second, and fifth columns shows the results from untreated platelet rich plasma (George King Plasma), untreated 99% immunodepleted Factor IX plasma, and untreated 90% immunodepleted Factor IX plasma and function as controls. Each plasma was either treated with free EACA (400 μg/mL) or fresh platelets loaded with EACA (5 μg/mL). The data demonstrate that free EACA had little to no effect on either the 99% (column 3) or the 90% (column 6) immunodepleted Factor IX plasmas' total thrombin generation. In contrast, fresh loaded platelets with EACA restored total thrombin generation to positive control concentrations in 90% immunodepleted Factor IX platelet rich plasma. No effect on total thrombin generation was measured when treating 99% immunodepleted Factor IX platelet rich plasma with EACA loaded platelets.

Example 7—Thrombin Generation in Congenital Hemophilia a and Congenital Hemophilia B

FIG. 18 shows that EACA loaded fresh platelets restored thrombin generation in 100%, 95%, and 90% congenital Factor VIII deficient (Hemophilia A) platelet rich plasma samples. All platelet rich plasma samples included 300,000 cell/μL. A positive George King Plasma sample was included, as well as, untreated 95% and 100% Factor VIII deficient platelet rich plasma samples (bottom two lines in FIG. 18). The three intervening line plots between the positive George King Plasma control and the untreated 90% and 95% Factor VIII deficient platelet rich plasma samples represent the fresh platelets loaded with EACA (4.7 μg/mL) ability to restore thrombin generation (nM) over time in 100%, 95%, and 90% congenital Factor VIII deficient platelet rich plasma samples. A greater increase in thrombin generation was measured in platelet rich samples that included some coagulation Factor VIII. For example, a higher concentration of thrombin was measured for 90% immunodepleted Factor VIII platelet rich plasma as compared to 95% immunodepleted Factor VIII platelet rich plasma. Similarly, a higher concentration of thrombin was measured for 95% immunodepleted Factor VIII platelet rich plasma as compared to 100% immunodepleted Factor VIII platelet rich plasma.

FIG. 19 shows that EACA loaded fresh platelets restored thrombin generation in 95% and 90% congenital Factor IX deficient (Hemophilia B) platelet rich plasma samples. All platelet rich plasma samples included 300,000 cell/μL. A positive George King Plasma sample was included, as well as, untreated 95% and 100% Factor VIX deficient platelet rich plasma samples (dotted lines in FIG. 19). No effect on thrombin generation was measured when fresh platelets loaded with EACA (4.7 μg/mL) were treated to 100% congenital Factor IX deficient platelet rich plasma. In contrast, fresh platelets loaded with EACA increased thrombin generation (nM) in both 95% and 90% congenital Factor IX deficient platelet rich plasma. A greater increase in thrombin generation was measured in platelet rich samples that included some coagulation Factor IX. For example, a higher concentration of thrombin was measured for 90% immunodepleted Factor IX platelet rich plasma as compared to 95% immunodepleted Factor IX platelet rich plasma.

The data in FIG. 20 show that fresh platelets loaded with EACA restored peak thrombin generation (nM) in 95% congenital Factor VIII deficient (Hemophilia A) platelet rich plasma and 95% congenital Factor IX deficient (Hemophilia B) platelet rich plasma. The first column is a positive George King Plasma control. Columns 3 and 4 show the effect of either free EACA (400 μg/mL) (column 3) or fresh platelets loaded with EACA (4.7 μg/mL)(column 4) on peak thrombin generation (nM) in 95% congenital Factor VIII deficient platelet rich plasma. The data show that fresh platelets loaded with EACA (4.7 μg/mL) significantly increased peak thrombin generation (column 4) relative to the free EACA (400 μg/mL)(column 3). Moreover, the significant increase in peak thrombin generation measured with the EACA loaded fresh platelets was achieved with about an 80 fold less concentration of EACA.

FIG. 20 also shows that fresh platelets loaded with EACA increased peak thrombin generation in 95% congenital Factor IX deficient platelet rich plasma more than free EACA alone. Columns 6 and 7 show the effect of either free EACA (400 μg/mL)(column 6) or fresh platelets loaded with EACA (4.7 μg/mL)(column 7) on peak thrombin generation in 95% congenital Factor IX deficient platelet rich plasma. The data show that fresh platelets loaded with EACA (4.7 μg/mL) significantly increased peak thrombin generation (column 7) relative to the free EACA at 400 μg/mL (column 6). Moreover, the significant increase in peak thrombin generation measured with the EACA loaded fresh platelets was achieved with about an 80 fold less concentration of EACA.

The data in FIG. 21 show that fresh platelets loaded with EACA restored total thrombin generation (nM) in 95% congenital Factor VIII deficient (Hemophilia A) platelet rich plasma and 95% congenital Factor IX deficient (Hemophilia B) platelet rich plasma. The first column is a positive George King Plasma control. Columns 3 and 4 show the effect of either free EACA (400 μg/mL) (column 3) or fresh platelets loaded with EACA (4.7 μg/mL)(column 4) on total thrombin generation (nM) in 95% congenital Factor VIII deficient platelet rich plasma. The data show that fresh platelets loaded with EACA (4.7 μg/mL) significantly increased total thrombin generation (column 4) relative to the free EACA (400 μg/mL)(column 3). Moreover, the significant increase in total thrombin generation measured with the EACA loaded fresh platelets was achieved with about an 80 fold less concentration of EACA.

FIG. 21 also shows that fresh platelets loaded with EACA increased total thrombin generation in 95% congenital Factor IX deficient platelet rich plasma more than free EACA alone. Columns 6 and 7 show the effect of either free EACA (400 μg/mL)(column 6) or fresh platelets loaded with EACA (4.7 μg/mL)(column 7) on total thrombin generation in 95% congenital Factor IX deficient platelet rich plasma. The data show that fresh platelets loaded with EACA (4.7 μg/mL) significantly increased total thrombin generation (column 7) relative to the free EACA at 400 μg/mL (column 6). Moreover, the significant increase in total thrombin generation measured with the EACA loaded fresh platelets was achieved with about an 80 fold less concentration of EACA.

Example 8 EACA-Loaded Thrombosomes in Congenital Hemophilia

A thrombin generation assay (TGA) stimulates and measures thrombin production. The TGA uses a coagulation stimulating agent combined with calcium to initiate coagulation and the resulting production of thrombin. To perform the TGA assay a first solution of 40 μL of thrombosomes in PBS, 40 μL of platelet rich plasma, and 20 μL of PPP-low were combined in triplicate wells. A PPP reagent stimulates a clotting cascade with low amounts of tissue factor and phospholipids. Additionally, a second solution of 40 μL of thrombosomes, 40 μL of platelet rich plasma, and 20 μL of a calibrator solution were combined in triplicate wells. The first and the second solution were combined at incubated at 37° C. for 10 minutes with 20 μL of Fluo-substrate (FluCa). A Fluo-substrate contains a fluorogenic substrate solubilized in DMSO (https://www.thrombinoscope.com/method-products/products/). After the 10 minute incubation the results were measured on a Thrombinoscope.

FIG. 22 shows thrombin generation (nM) of varying concentrations of EACA loaded thrombosomes. The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in George King Plasma. The EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³ mg/thrombosome. The data demonstrate a correlation between increased EACA loaded thrombosome concentration and increased thrombin generation (nM). That is, a higher concentration of EACA loaded thrombosomes generated higher concentration of thrombin.

FIG. 23 shows EACA loaded thrombosomes of varying concentrations generate thrombin (nM) in 95% congenital Factor IX deficient (Hemophilia B) platelet rich plasma. The data show that generally, higher concentrations of EACA loaded thrombosomes are able to generate increased concentrations of thrombin. The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95% congenital Factor IX deficient platelet rich plasma and the EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³. However, there may be a limit to the increase in thrombin generation. For example, the concentration of EACA loaded thrombosomes that resulted in the highest concentration of thrombin was 150 k/μL. However, thrombin generation with the EACA loaded thrombosomes at 300 k/μL resulted in a thrombin concentration less than both EACA loaded thrombosomes at 37.5 k/μL and 75 k/μL.

The data in FIG. 24 show that EACA loaded thrombosomes restored peak thrombin generation in 95% congenital Factor IX deficient (Hemophilia B) platelet rich plasma. The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95% congenital Factor IX deficient platelet rich plasma and the EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³ Again, there may be a limit in the increase of peak thrombin generation from EACA loaded thrombosomes. For example, the concentration of EACA loaded thrombosomes that resulted in the highest peak thrombin generation concentration was 150 k/μL.

The data in FIG. 25 show that EACA loaded thrombosomes restored total thrombin generation in 95% congenital Factor IX deficient (Hemophilia B) platelet rich plasma. The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95% congenital Factor IX deficient platelet rich plasma and the EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³ Again, there may be a limit in the increase of total thrombin generation from EACA loaded thrombosomes. For example, the concentration of EACA loaded thrombosomes that resulted in the highest peak thrombin generation concentration was 75 k/μL.

The data in FIG. 26 show that EACA loaded thrombosomes restored thrombin generation (total) in 95% congenital Factor IX deficient (Hemophilia B) platelet rich plasma and 95% immunodepleted Factor IX deficient (Hemophilia B) platelet rich plasma. The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95% congenital Factor IX deficient platelet rich plasma and the EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³. The data show that EACA loaded thrombosomes of a sufficient concentration restored total thrombin generation in 95% immunodepleted Factor IX deficient platelet rich plasma to approximately positive control concentrations (George King Plasma) and significant more than unloaded thrombosomes in 95% immunodepleted Factor IX deficient platelet rich plasma. Similarly, EACA loaded thrombosomes restored total thrombin generation (nM) in congenital Factor IX deficient platelet rich plasma significantly more than unloaded thrombosomes in 95% congenital Factor IX deficient plate rich plasma. Again, there may be a limit in the increase of total thrombin generation from EACA loaded thrombosomes in both 95% immunodepleted and congenital Factor IX deficient platelet rich plasma. For example, the box shown in FIG. 25 highlight a concentration range of EACA loaded thrombosomes that resulted in the highest total thrombin generation concentration measured. Without wishing to be limited, the data suggest a concentration range of EACA loaded thrombosomes of between about 50 k/μL and 150 k/μL result in the highest concentrations of thrombin measured.

FIG. 27 shows thrombin generation (nM) of varying concentrations of EACA loaded thrombosomes. The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95% congenital Factor VIII deficient (Hemophilia A) platelet rich plasma. The EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³ mg/thrombosome. The data demonstrate a correlation between increased EACA loaded thrombosome concentration and increased thrombin generation (nM). That is, a higher concentration of EACA loaded thrombosomes generated higher concentration of thrombin.

The data in FIG. 28 show that EACA loaded thrombosomes restored peak thrombin generation in 95% congenital Factor VIII deficient (Hemophilia A) platelet rich plasma to positive control concentrations (George King Plasma). The EACA loaded thrombosome concentrations tested included 0 k/μL, 4.7 k/μL, 9.4 k/μL, 18.8 k/μL, 37.5 k/μL, 75 k/μL, 150 k/μL, and 300 k/μL in 95% congenital Factor VIII deficient platelet rich plasma and the EACA loaded thrombosomes contained EACA at a concentration of 2.2×10⁻¹³. There may be a limit in the increase of peak thrombin generation from EACA loaded thrombosomes. For example, the concentration of EACA loaded thrombosomes that resulted in the highest peak thrombin generation concentration was 150 k/μL.

Embodiments

Embodiment 1 is a method of treating congenital Hemophilia A in a subject, wherein the subject is a subject in need thereof, the method comprising: administering a therapeutically effective amount of anti-fibrinolytic loaded platelets to the subject in need thereof.

Embodiment 2 is a method of treating congenital Hemophilia B in a subject, wherein the subject is a subject in need thereof, the method comprising: administering a therapeutically effective amount of anti-fibrinolytic loaded platelets to the subject in need thereof.

Embodiment 3 is the method of any one of embodiments 1-2, wherein the concentration of the therapeutically effective amount of anti-fibrinolytic loaded into the platelets is from about 100 μM to about 10 mM.

Embodiment 4 is a method of treating congenital Hemophilia A in a subject, wherein the subject is a subject in need thereof, the method comprising, administering a therapeutically effective amount of loaded thrombosomes to a subject in need thereof.

Embodiment 5 is a method of treating congenital Hemophilia B in a subject, wherein the subject is a subject in need thereof, the method comprising, administering a therapeutically effective amount of loaded thrombosomes to a subject in need thereof.

Embodiment 6 is the method of embodiment 4 or 5, wherein the loaded thrombosomes are loaded with an anti-fibrinolytic.

Embodiment 7 is the method of embodiment of 5 or 6, wherein the anti-fibrinolytic is selected from the group consisting of ε-aminocaproic acid, aprotinin, aminomethylbenzoic acid, tranexamic acid, and fibrinogen.

Embodiment 8 is the method of embodiment 7, wherein the anti-fibrinolytic is F-aminocaproic acid.

Embodiment 9 is the method of embodiment 7 or 8, wherein F-aminocaproic acid is present in a concentration from about 1 μM to about 100 mM.

Embodiment 10 is a method of treating congenital hemophilia A in a subject, wherein the subject is a subject in need thereof, the method comprising:

administering a therapeutically effective amount of unloaded thrombosomes to the subject in need thereof.

Embodiment 11 is a method of treating congenital hemophilia B in a subject, wherein the subject is a subject in need thereof, the method comprising:

administering a therapeutically effective amount of unloaded thrombosomes to the subject in need thereof.

Embodiment 12 is the method of embodiment 10 or 11, wherein the concentration of the therapeutically effective amount of unloaded thrombosomes is from about 1×10² particles/kg to about 1×10¹³ particles/kg.

Embodiment 13 is a method of treating a coagulopathy in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition comprising platelets or platelet derivatives and an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent.

Embodiment 14 is a method of treating a coagulopathy in a subject, wherein the subject is a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a composition prepared by a process comprising incubating platelets with an incubating agent comprising one or more salts, a buffer, optionally a cryoprotectant, and optionally an organic solvent, to form the composition.

Embodiment 15 is the method of embodiment 13 or 14, wherein the composition is administered following administration to the subject an antiplatelet agent or an anticoagulant or a subject having congenital hemophilia.

Embodiment 16 is a method of treating congenital hemophilia in a subject in need thereof, comprising administering to the subject anti-fibrinolytic loaded platelets, cryopreserved platelets, and/or freeze-dried platelets according to Embodiment 1 or 2, wherein following administration peak thrombin increases by at least 25%.

Embodiment 17 is a method of treating congenital hemophilia in a subject in need thereof, comprising administering to the subject anti-fibrinolytic loaded platelets, cryopreserved platelets, and/or freeze-dried platelets according to Embodiment 1 or 2, wherein following administration peak thrombin increases by at least 40%.

Embodiment 18 is a method of treating congenital hemophilia in a subject in need thereof, comprising administering to the subject anti-fibrinolytic loaded platelets, cryopreserved platelets, and/or freeze-dried platelets according to Embodiment 1 or 2, wherein following administration ETP increases by at least 40%. 

1. A method of treating congenital Hemophilia in a subject, the method comprising: administering a therapeutically effective amount of anti-fibrinolytic loaded platelets and/or a therapeutically effective amount of anti-fibrinolytic loaded thrombosomes to the subject in need thereof.
 2. (canceled)
 3. The method of claim 1, wherein the congenital Hemophilia is congenital Hemophilia A.
 4. The method of claim, wherein the congenital Hemophilia is congenital Hemophilia B.
 5. The method of claim 1, wherein the concentration of the therapeutically effective amount of an anti-fibrinolytic loaded into the platelets is from about 100 μM to about 10 mM.
 6. The method of claim 5, wherein the concentration of the therapeutically effective amount of the anti-fibrinolytic loaded into the platelets is from about 500 μM to about 5 mM.
 7. The method of claim 6, wherein the concentration of the therapeutically effective amount of the anti-fibrinolytic loaded into the platelets is from about 1 mM to about 3 mM.
 8. The method of claim 1, wherein the concentration of the therapeutically effective amount of an anti-fibrinolytic loaded into the platelets is about 500 μM to about 10 mM.
 9. A method of treating congenital Hemophilia in a subject, the method comprising: administering a therapeutically effective amount of anti-fibrinolytic loaded cryopreserved platelets to the subject in need thereof.
 10. The method of claim 9, wherein the congenital Hemophilia is congenital Hemophilia A.
 11. The method of claim 9, wherein the congenital Hemophilia is congenital Hemophilia B.
 12. The method of claim 9, wherein the concentration of the therapeutically effective amount of anti-fibrinolytic loaded cryopreserved platelets or freeze-dried platelets is from about 1×10² particles/kg to about 1×10¹³ particles/kg.
 13. The method of claim 12, wherein the concentration of the therapeutically effective amount of anti-fibrinolytic loaded cryopreserved platelets or freeze-dried platelets is from about 1×10⁴ particles/kg to about 1×10¹¹ particles/kg.
 14. The method of claim 13, wherein the concentration of the therapeutically effective amount of anti-fibrinolytic loaded cryopreserved platelets or freeze-dried platelets is from about 1×10⁶ particles/kg to about 1×10⁹ particles/kg.
 15. The method of claim 9, wherein the concentration of the therapeutically effective amount of anti-fibrinolytic loaded cryopreserved platelets or freeze-dried platelets is at least 1×10⁴ particles/kg.
 16. The method of claim 1, wherein the anti-fibrinolytic is selected from the group consisting of ε-aminocaproic acid, aprotinin, aminomethylbenzoic acid, tranexamic acid, and fibrinogen.
 17. The method of claim 16, wherein the anti-fibrinolytic is F-aminocaproic acid.
 18. A method of treating congenital Hemophilia A or congenital Hemophilia B, comprising administering a therapeutically effective amount of anti-fibrinolytic loaded platelets, wherein the anti-fibrinolytic loaded platelets are prepared by a process comprising contacting platelets with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets.
 19. A method to treat congenital Hemophilia A or congenital Hemophilia B comprising A) pooling platelets from a plurality of donors, B) contacting the platelets from step (A) with an anti-fibrinolytic and with a loading buffer including a salt, a base, a loading agent, and optionally at least one organic solvent, to form the anti-fibrinolytic loaded platelets; and C) administering the anti-fibrinolytic loaded platelets to a subject in need thereof.
 20. The method of claim 1, wherein administering comprises administering topically, intravenously, intramuscularly, and combinations thereof.
 21. The method of claim 1, wherein the method does not comprise administering an anti-fibrinolytic. 